Formulations with reduced oxidation

ABSTRACT

The invention provides formulations comprising a protein in combination with a compound that prevents oxidation of the protein. The invention also provides methods for making such formulations and methods of using such formulations. The invention further provides methods of screening for compounds that prevent oxidation of a protein in a protein composition and methods of preventing oxidation of a protein in a formulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/207,911, filed Mar. 13, 2014, which claims thebenefit of U.S. Provisional Application No. 61/780,852, filed Mar. 13,2013, the contents of both of which are hereby incorporated by referencein their entirety.

FIELD OF THE INVENTION

This invention relates to formulations comprising a protein and furthercomprising a compound that prevents oxidation of said protein, methodsfor producing and using the formulations as well as methods of screeningfor compounds that prevent protein oxidation in protein compositions.

BACKGROUND OF THE INVENTION

Oxidative degradation of amino acid residues is a commonly observedphenomenon in protein pharmaceuticals. A number of amino acid residuesare susceptible to oxidation, particularly methionine (Met), cysteine(Cys), histidine (His), tryptophan (Trp), and tyrosine (Tyr) (Li et al.,Biotechnology and Bioengineering 48:490-500 (1995)). Oxidation istypically observed when the protein is exposed to hydrogen peroxide,light, metal ions or a combination of these during various processingsteps (Li et al., Biotechnology and Bioengineering 48:490-500 (1995)).In particular, proteins exposed to light (Wei, et al., AnalyticalChemistry 79(7):2797-2805 (2007)), AAPH or Fenton reagents (Ji et al., JPharm Sci 98(12):4485-500 (2009)) have shown increased levels ofoxidation on tryptophan residues, whereas those exposed to hydrogenperoxide have typically shown only methionine oxidation (Ji et al., JPharm Sci 98(12):4485-500 (2009)). Light exposure can result in proteinoxidation through the formation of reactive oxygen species (ROS)including singlet oxygen, hydrogen peroxide and superoxide (Li et al.,Biotechnology and Bioengineering 48:490-500 (1995); Wei, et al.,Analytical Chemistry 79(7):2797-2805 (2007); Ji et al., J Pharm Sci98(12):4485-500 (2009); Frokjaer et al., Nat Rev Drug Discov4(4):298-306 (2005)), whereas protein oxidation typically occurs viahydroxyl radicals in the Fenton mediated reaction (Prousek et al., Pureand Applied Chemistry 79(12):2325-2338 (2007)) and via alkoxyl peroxidesin the AAPH mediated reaction (Werber et al., J Pharm Sci 100(8):3307-15(2011)). Oxidation of tryptophan leads to a myriad of oxidationproducts, including hydroxytryptophan, kynurenine, andN-formylkynurenine, and has the potential to impact safety and efficacy(Li et al., Biotechnology and Bioengineering 48:490-500 (1995); Ji etal., J Pharm Sci 98(12):4485-500 (2009); Frokjaer et al., Nat Rev DrugDiscov 4(4):298-306 (2005)). Oxidation of a particular tryptophanresidue in the heavy chain complementarity determining region (CDR) of amonoclonal antibody that correlated to loss of biological function hasbeen reported (Wei, et al., Analytical Chemistry 79(7):2797-2805(2007)). Trp oxidation mediated by a histidine coordinated metal ion hasrecently been reported for a Fab molecule (Lam et al., Pharm Res28(10):2543-55 (2011)). Autoxidation of polysorbate 20 in the Fabformulation, leading to the generation of various peroxides, has alsobeen invoked in the same report. Autoxidation-induced generation ofthese peroxides can also lead to methionine oxidation in the proteinduring long-term storage of the drug product since Met residues inproteins have been suggested to act as internal antioxidants (Levine etal., Proceedings of the National Academy of Sciences of the UnitedStates of America 93(26):15036-15040 (1996)) and are easily oxidized byperoxides. Oxidation of amino acid residues has the potential to impactthe biological activity of the protein. This may be especially true formonoclonal antibodies (mAbs). Methionine oxidation at Met254 and Met430in an IgG1 mAb potentially impacts serum half-life in transgenic mice(Wang et al., Molecular Immunology 48(6-7):860-866 (2011)) and alsoimpacts binding of human IgG1 to FcRn and Fc-gamma receptors(Bertolotti-Ciarlet et al., Molecular Immunology 46(8-9)1878-82 (2009)).

The stability of proteins, especially in liquid state, needs to beevaluated during drug product manufacturing and storage. The developmentof pharmaceutical formulations sometimes includes addition ofantioxidants to prevent oxidation of the active ingredient. Addition ofL-methionine to formulations has resulted in reduction of methionineresidue oxidation in proteins and peptides (Ji et al., J Pharm Sci98(12):4485-500 (2009); Lam et al., Journal of Pharmaceutical Sciences86(11):1250-1255 (1997)) Likewise, addition of L-tryptophan has beenshown to reduce oxidation of tryptophan residues (Ji et al., J Pharm Sci98(12):4485-500 (2009); Lam et al., Pharm Res 28(10):2543-55 (2011)).L-Trp, however, possesses strong absorbance in the UV region (260-290nm) making it a primary target during photo-oxidation (Creed, D.,Photochemistry and Photobiology 39(4):537-562 (1984)). Trp has beenhypothesized as an endogenous photosensitizer enhancing the oxygendependent photo-oxidation of tyrosine (Babu et al., Indian J BiochemBiophys 29(3):296-8 (1992)) and other amino acids (Bent et al., Journalof the American Chemical Society 97(10):2612-2619 (1975)). It has beendemonstrated that L-Trp can generate hydrogen peroxide when exposed tolight and that L-Trp under UV light produces hydrogen peroxide via thesuperoxide anion (McCormick et al., Science 191(4226):468-9 (1976);Wentworth et al., Science 293(5536):1806-11 (2001); McCormick et al.,Journal of the American Chemical Society 100:312-313 (1978)).Additionally, tryptophan is known to produce singlet oxygen uponexposure to light (Davies, M. J., Biochem Biophys Res Commun305(3):761-70 (2003)). Similar to the protein oxidation induced byautoxidation of polysorbate 20, it is possible that protein oxidationcan occur upon ROS generation by other excipients in the proteinformulation (e.g. L-Trp) under normal handling conditions.

It is apparent from recent studies that the addition of standardexcipients, such as L-Trp and polysorbates, to protein compositions thatare meant to stabilize the protein can result in unexpected andundesired consequences such as ROS-induced oxidation of the protein.Therefore, there remains a need for the identification of alternativeexcipients for use in protein compositions and the development of suchcompositions.

BRIEF SUMMARY OF THE INVENTION

Provided herein are formulations comprising a protein and a compoundthat prevents oxidation of the protein in the formulation, methods ofmaking the formulations, and methods of screening compounds that preventoxidation of a protein in a protein formulation.

In one aspect, provided herein is a formulation comprising a protein anda compound which prevents oxidation of the protein in the formulation,wherein the compound is of formula:

wherein R² is selected from hydrogen, hydroxyl, —COOH, and —CH₂COOH;

R³ is selected from hydrogen, hydroxyl, —COOH, —CH₂COOH, and—CH₂CHR^(3a)(NH₂); wherein R^(3a) is COOH or hydrogen;

R⁴, R⁵, R⁶, and R⁷ are independently selected from hydrogen andhydroxyl;

provided that one of R², R³, R⁴, R⁵, R⁶, and R⁷ is hydroxyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of formula:

wherein R² and R³ are independently selected from hydrogen, hydroxyl,—COOH, and —CH₂COOH; and

R⁴, R⁵, R⁶, and R⁷ are independently selected from hydrogen andhydroxyl;

provided that one of R², R³, R⁴, R⁵, R⁶, and R⁷ is hydroxyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of formula:

wherein R^(3a) is COOH or hydrogen;

R², R⁴, R⁵, R⁶, and R⁷ are independently selected from hydrogen andhydroxyl, provided that one of R², R⁴, R⁵, R⁶, and R⁷ is hydroxyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments, R⁴, R⁵ or R⁷ in any of the formula above ishydroxyl. In some embodiments, the compound is selected from the groupconsisting of 5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxy indole,and serotonin.

In some embodiments, the formulation is a liquid formulation. In someembodiments, the formulation is a pharmaceutical formulation suitablefor administration to a subject. In some embodiments, the formulation isaqueous.

In some embodiments, the compound in the formulation is from about 0.3mM to about 1 mM. In some embodiments, the compound prevents oxidationof tryptophan, cysteine, histidine, tyrosine, and/or methionine in theprotein. In some embodiments, the compound prevents oxidation of theprotein by a reactive oxygen species. In some embodiments, the reactiveoxygen species is selected from the group consisting of singlet oxygen,hydrogen peroxide, a hydroxyl radical, and an alkyl peroxide.

In some embodiments, the protein is susceptible to oxidation. In someembodiments, a tryptophan amino acid in the protein is susceptible tooxidation. In some embodiments, the protein is an antibody (e.g., apolyclonal antibody, a monoclonal antibody, a humanized antibody, ahuman antibody, a chimeric antibody, or antibody fragment). In someembodiments, the protein concentration in the formulation is about 1mg/mL to about 250 mg/mL.

In some embodiments, the formulation further comprises one or moreexcipients selected from the group consisting of a stabilizer, a buffer,a surfactant, and a tonicity agent. In some embodiments, the formulationhas a pH of about 4.5 to about 7.0.

In another aspect, provided herein is a method of making a proteinformulation (such as a liquid formulation) comprising adding an amountof a compound that prevents oxidation of a protein to the formulation,wherein the compound is of formula:

wherein R² is selected from hydrogen, hydroxyl, —COOH, and —CH₂COOH;

R³ is selected from hydrogen, hydroxyl, —COOH, —CH₂COOH, and—CH₂CHR^(3a)(NH₂); wherein R^(3a) is COOH or hydrogen;

R⁴, R⁵, R⁶, and R⁷ are independently selected from hydrogen andhydroxyl;

provided that one of R², R³, R⁴, R⁵, R⁶, and R⁷ is hydroxyl;

or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of preventing oxidationof a protein in a protein formulation (such as a liquid formulation)comprising adding an amount of a compound that prevents oxidation of aprotein to the formulation, wherein the compound is of formula:

wherein R² is selected from hydrogen, hydroxyl, —COOH, and —CH₂COOH;

R³ is selected from hydrogen, hydroxyl, —COOH, —CH₂COOH, and—CH₂CHR^(3a)(NH₂); wherein R^(3a) is COOH or hydrogen;

R⁴, R⁵, R⁶, and R⁷ are independently selected from hydrogen andhydroxyl;

provided that one of R², R³, R⁴, R⁵, R⁶, and R⁷ is hydroxyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments of the methods described herein, the compound is acompound of formula:

wherein R² and R³ are independently selected from hydrogen, hydroxyl,—COOH, and —CH₂COOH; and

R⁴, R⁵, R⁶, and R⁷ are independently selected from hydrogen andhydroxyl;

provided that one of R², R³, R⁴, R⁵, R⁶, and R⁷ is hydroxyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments of the methods described herein, the compound is acompound of formula:

wherein R^(3a) is COOH or hydrogen;

R², R⁴, R⁵, R⁶, and R⁷ are independently selected from hydrogen andhydroxyl, provided that one of R², R⁴, R⁵, R⁶, and R⁷ is hydroxyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments, R⁴, R⁵ or R⁷ in any of the formula above ishydroxyl. In some embodiments, the compound is selected from the groupconsisting of 5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxy indole,and serotonin.

In some embodiments, the formulation is a liquid formulation. In someembodiments, the formulation is a pharmaceutical formulation suitablefor administration to a subject. In some embodiments, the formulation isaqueous. In some embodiments, the compound in the formulation is fromabout 0.3 mM to about 1 mM.

In some embodiments, the compound prevents oxidation of tryptophan,cysteine, histidine, tyrosine, and/or methionine in the protein. In someembodiments, the compound prevents oxidation of the protein by areactive oxygen species. In some embodiments, the reactive oxygenspecies is selected from the group consisting of singlet oxygen,hydrogen peroxide, a hydroxyl radical, and an alkyl peroxide.

In some embodiments, the protein is susceptible to oxidation. In someembodiments, a tryptophan amino acid in the protein is susceptible tooxidation. In some embodiments, the protein is an antibody (e.g., apolyclonal antibody, a monoclonal antibody, a humanized antibody, ahuman antibody, a chimeric antibody, or antibody fragment). In someembodiments, the protein concentration in the formulation is about 1mg/mL to about 250 mg/mL.

In some embodiments, the formulation further comprises one or moreexcipients selected from the group consisting of a stabilizer, a buffer,a surfactant, and a tonicity agent. In some embodiments, the formulationhas a pH of about 4.5 to about 7.0.

In another aspect, provided herein is a method of screening a compoundthat prevents oxidation of a protein in a protein composition,comprising selecting a compound that has lower oxidation potential andless photosensitivity as compared to L-tryptophan, and testing theeffect of the selected compound on preventing oxidation of the protein.

In some embodiments, the photosensitivity is measured based on theamount of H₂O₂ produced by the compound upon light exposure. In someembodiments, the compound that produces less than about 10% of theamount of H₂O₂ produced by L-tryptophan is selected. In someembodiments, the oxidation potential is measured by cyclic voltammetry.In some embodiments, the selected compound is tested for the effect onpreventing oxidation of the protein by reactive oxygen species generatedby 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), light, and/or aFenton reagent.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art. These andother embodiments of the invention are further described by the detaileddescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of graphs demonstrating the oxidation of A) Fab inmAb1, and B) Fc in mAb1 after eight hours of light exposure at 250 W/m².mAb1 was present at 5 mg/mL in 20 mM histidine acetate, 250 mMtrehalose, 0.02% polysorbate 20. All vials were placed in the lightboxexcept the mAb1 Ref Mat. Foil CTRL vials were covered in foil beforeplacement in the lightbox. Three separate experimental vials wereaveraged for each sample, except “10 mM Met, 1 mM Trp” (*) was theaverage of two experimental vials, and mAb1 Ref Mat was one experimentalvial with three independent injections on the HPLC. Error bars representone standard deviation.

FIG. 2 is a graph showing dose dependent H₂O₂ production by L-Trp.Diamonds indicate L-Trp alone; Triangles indicate L-Trp+SOD; Circles andSquares indicate L-Trp+NaN₃±SOD. All studies were performed in 20 mML-His HCl, pH 5.5.

FIG. 3 is a series of graphs demonstrating A) Hydrogen peroxide (H₂O₂)production in 50 mg/mL mAb1 formulations containing 3.2 mM L-Trp whenexposed to ambient light conditions for 1, 3 and 7 days and B) Percent(%) Fab oxidation in mAb1 formulations containing 3.2 mM L-Trp after 10days of exposure to ambient light conditions.

FIG. 4 is a series of graphs showing hydrogen peroxide generation bytryptophan derivatives and indole derivatives under light stress for 4hours at 250 W/m². A) Screening of tryptophan derivatives (1 mM) forhydrogen peroxide (μM) generation in a 20 mM HisAc pH5.5 formulation. B)Screening of indole derivatives (1 mM) for hydrogen peroxide (μM)generation in a 20 mM HisAc pH5.5 formulation.

FIG. 5 is a graph showing the effect of NaN₃ on H₂O₂ production byvarious Trp derivatives upon light exposure. Data is shown as a ratiowith respect to peroxide generated by L-Trp.

FIG. 6 is a graph showing the correlation between oxidation potentialand light-induced peroxide formation. The boxed region shows candidateantioxidant compounds.

FIG. 7 is a series of graphs showing the oxidation of A) Fab in mAb1,and B) Fc in mAb1 after AAPH incubation. All samples were incubated withAAPH except mAb1 Ref Mat and No AAPH. All samples were incubated at 40°C. except mAb1 Ref Mat. Data shown are the average of three experimentalsamples±1SD, except mAb1 Ref Mat which is the average of six HPLCinjections without error bars.

FIG. 8 is series of graphs showing the oxidation of A) Fab in mAb1, andB) Fc in mAb1 after sixteen hours of light exposure at 250 W/m². Allvials were placed in the lightbox except the mAb1 Ref Mat. Foil CTRLvials were covered in foil before placement in the lightbox. Threeseparate experimental vials were averaged for each sample, exceptL-tryptophanamide (*) was the average of two experimental vials and mAb1Ref Mat was one vial with three independent injections on the HPLC.Error bars represent one standard deviation.

DETAILED DESCRIPTION I. Definitions

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations are sterile.

A “sterile” formulation is aseptic or free or essentially free from allliving microorganisms and their spores.

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage. Preferably, the formulationessentially retains its physical and chemical stability, as well as itsbiological activity upon storage. The storage period is generallyselected based on the intended shelf-life of the formulation. Variousanalytical techniques for measuring protein stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example.Stability can be measured at a selected amount of light exposure and/ortemperature for a selected time period. Stability can be evaluatedqualitatively and/or quantitatively in a variety of different ways,including evaluation of aggregate formation (for example using sizeexclusion chromatography, by measuring turbidity, and/or by visualinspection); evaluation of ROS formation (for example by using a lightstress assay or a 2,2′-Azobis(2-Amidinopropane)Dihydrochloride (AAPH)stress assay); oxidation of specific amino acid residues of the protein(for example a Trp residue and/or a Met residue of a monoclonalantibody); by assessing charge heterogeneity using cation exchangechromatography, image capillary isoelectric focusing (icIEF) orcapillary zone electrophoresis; amino-terminal or carboxy-terminalsequence analysis; mass spectrometric analysis; SDS-PAGE analysis tocompare reduced and intact antibody; peptide map (for example tryptic orLYS-C) analysis; evaluating biological activity or target bindingfunction of the protein (e.g., antigen binding function of an antibody);etc. Instability may involve any one or more of: aggregation,deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation and/orTrp oxidation), isomerization (e.g. Asp isomeriation),clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation),succinimide formation, unpaired cysteine(s), N-terminal extension,C-terminal processing, glycosylation differences, etc.

A protein “retains its physical stability” in a pharmaceuticalformulation if it shows no signs or very little of aggregation,precipitation and/or denaturation upon visual examination of colorand/or clarity, or as measured by UV light scattering or by sizeexclusion chromatography.

A protein “retains its chemical stability” in a pharmaceuticalformulation, if the chemical stability at a given time is such that theprotein is considered to still retain its biological activity as definedbelow. Chemical stability can be assessed by detecting and quantifyingchemically altered forms of the protein. Chemical alteration may involveprotein oxidation which can be evaluated using tryptic peptide mapping,reverse-phase high-performance liquid chromatography (HPLC) and liquidchromatography-mass spectrometry (LC/MS), for example. Other types ofchemical alteration include charge alteration of the protein which canbe evaluated by ion-exchange chromatography or icIEF, for example.

A protein “retains its biological activity” in a pharmaceuticalformulation, if the biological activity of the protein at a given timeis within about 10% (within the errors of the assay) of the biologicalactivity exhibited at the time the pharmaceutical formulation wasprepared as determined for example in an antigen binding assay for amonoclonal antibody.

As used herein, “biological activity” of a protein refers to the abilityof the protein to bind its target, for example the ability of amonoclonal antibody to bind to an antigen. It can further include abiological response which can be measured in vitro or in vivo. Suchactivity may be antagonistic or agonistic.

A protein which is “susceptible to oxidation” is one comprising one ormore residue(s) that has been found to be prone to oxidation such as,but not limited to, methionine (Met), cysteine (Cys), histidine (His),tryptophan (Trp), and tyrosine (Tyr). For example, a tryptophan aminoacid in the Fab portion of a monoclonal antibody or a methionine aminoacid in the Fc portion of a monoclonal antibody may be susceptible tooxidation.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer of this invention preferably has a pH in the range from about 4.5to about 8.0. For example, histidine acetate is an example of a bufferthat will control the pH in this range.

A “preservative” is a compound which can be optionally included in theformulation to essentially reduce bacterial action therein, thusfacilitating the production of a multi-use formulation, for example.Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (amixture of alkylbenzyldimethylammonium chlorides in which the alkylgroups are long-chain compounds), and benzethonium chloride. Other typesof preservatives include aromatic alcohols such as phenol, butyl andbenzyl alcohol, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. In oneembodiment, the preservative herein is benzyl alcohol.

As used herein, a “surfactant” refers to a surface-active agent,preferably a nonionic surfactant. Examples of surfactants herein includepolysorbate (for example, polysorbate 20 and, polysorbate 80); poloxamer(e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodiumlaurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-,or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.); polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc.); etc. In oneembodiment, the surfactant herein is polysorbate 20.

“Pharmaceutically acceptable” excipients or carriers as used hereininclude pharmaceutically acceptable carriers, stabilizers, buffers,acids, bases, sugars, preservatives, surfactants, tonicity agents, andthe like, which are well known in the art (Remington: The Science andPractice of Pharmacy, 22^(nd) Ed., Pharmaceutical Press, 2012). Examplesof pharmaceutically acceptable excipients include buffers such asphosphate, citrate, acetate, and other organic acids; antioxidantsincluding ascorbic acid, L-tryptophan and methionine; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; metalcomplexes such as Zn-protein complexes; chelating agents such as EDTA:sugar alcohols such as mannitol or sorbitol; salt-forming counterionssuch as sodium; and/or nonionic surfactants such as polysorbate,poloxamer, polyethylene glycol (PEG), and PLURONICS™. “Pharmaceuticallyacceptable” excipients or carriers are those which can reasonably beadministered to a subject to provide an effective dose of the activeingredient employed and that are nontoxic to the subject being exposedthereto at the dosages and concentrations employed.

The protein which is formulated is preferably essentially pure anddesirably essentially homogeneous (e.g., free from contaminatingproteins etc.). “Essentially pure” protein means a compositioncomprising at least about 90% by weight of the protein (e.g., monoclonalantibody), based on total weight of the composition, preferably at leastabout 95% by weight. “Essentially homogeneous” protein means acomposition comprising at least about 99% by weight of the protein(e.g., monoclonal antibody), based on total weight of the composition.

The terms “protein” “polypeptide” and “peptide” are used interchangeablyherein to refer to polymers of amino acids of any length. The polymermay be linear or branched, it may comprise modified amino acids, and itmay be interrupted by non-amino acids. The terms also encompass an aminoacid polymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, proteins containing one or more analogs ofan amino acid (including, for example, unnatural amino acids, etc.), aswell as other modifications known in the art. Examples of proteinsencompassed within the definition herein include mammalian proteins,such as, e.g., renin; a growth hormone, including human growth hormoneand bovine growth hormone; growth hormone releasing factor; parathyroidhormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;insulin A-chain; insulin B-chain; proinsulin; follicle stimulatinghormone; calcitonin; luteinizing hormone; glucagon; leptin; clottingfactors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; a tumor necrosis factor receptor such as deathreceptor 5 and CD120; TNF-related apoptosis-inducing ligand (TRAIL);B-cell maturation antigen (BCMA); B-lymphocyte stimulator (BLyS); aproliferation-inducing ligand (APRIL); enkephalinase; RANTES (regulatedon activation normally T-cell expressed and secreted); human macrophageinflammatory protein (MIP-1-alpha); a serum albumin such as human serumalbumin; Muellerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbialprotein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyteassociated antigen (CTLA), such as CTLA-4; inhibin; activin;platelet-derived endothelial cell growth factor (PD-ECGF); a vascularendothelial growth factor family protein (e.g., VEGF-A, VEGF-B, VEGF-C,VEGF-D, and P1GF); a platelet-derived growth factor (PDGF) familyprotein (e.g., PDGF-A, PDGF-B, PDGF-C, PDGF-D, and dimers thereof);fibroblast growth factor (FGF) family such as aFGF, bFGF, FGF4, andFGF9; epidermal growth factor (EGF); receptors for hormones or growthfactors such as a VEGF receptor(s) (e.g., VEGFR1, VEGFR2, and VEGFR3),epidermal growth factor (EGF) receptor(s) (e.g., ErbB1, ErbB2, ErbB3,and ErbB4 receptor), platelet-derived growth factor (PDGF) receptor(s)(e.g., PDGFR-α and PDGFR-β), and fibroblast growth factor receptor(s);TIE ligands (Angiopoietins, ANGPT1, ANGPT2); Angiopoietin receptor suchas TIE1 and TIE2; protein A or D; rheumatoid factors; a neurotrophicfactor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3,-4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor suchas NGF-b; transforming growth factor (TGF) such as TGF-alpha andTGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5;insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I(brain IGF-I), insulin-like growth factor binding proteins (IGFBPs); CDproteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin;osteoinductive factors; immunotoxins; a bone morphogenetic protein(BMP); a chemokine such as CXCL12 and CXCR4; an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; a cytokine such as interleukins (ILs),e.g., IL-1 to IL-10; midkine; superoxide dismutase; T-cell receptors;surface membrane proteins; decay accelerating factor; viral antigen suchas, for example, a portion of the AIDS envelope; transport proteins;homing receptors; addressins; regulatory proteins; integrins such asCD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; ephrins; Bv8;Delta-like ligand 4 (DLL4); Del-1; BMP9; BMP10; Follistatin; Hepatocytegrowth factor (HGF)/scatter factor (SF); Alk1; Robo4; ESM1; Perlecan;EGF-like domain, multiple 7 (EGFL7); CTGF and members of its family;thrombospondins such as thrombospondinl and thrombospondin2; collagenssuch as collagen IV and collagen XVIII; neuropilins such as NRP1 andNRP2; Pleiotrophin (PTN); Progranulin; Proliferin; Notch proteins suchas Notch1 and Notch4; semaphorins such as Sema3A, Sema3C, and Sema3F; atumor associated antigen such as CA125 (ovarian cancer antigen);immunoadhesins; and fragments and/or variants of any of the above-listedproteins as well as antibodies, including antibody fragments, binding toone or more protein, including, for example, any of the above-listedproteins.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity.

An “isolated” protein (e.g., an isolated antibody) is one which has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials which would interfere with research, diagnostic ortherapeutic uses for the protein, and may include enzymes, hormones, andother proteinaceous or nonproteinaceous solutes. Isolated proteinincludes the protein in situ within recombinant cells since at least onecomponent of the protein's natural environment will not be present.Ordinarily, however, isolated protein will be prepared by at least onepurification step.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “constant domain” refers to the portion of an immunoglobulinmolecule having a more conserved amino acid sequence relative to theother portion of the immunoglobulin, the variable domain, which containsthe antigen binding site. The constant domain contains the C_(H)1,C_(H)2 and C_(H)3 domains (collectively, CH) of the heavy chain and theCHL (or CL) domain of the light chain.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “V_(H).” Thevariable domain of the light chain may be referred to as “V_(L).” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions (HVRs) both in thelight-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any mammalianspecies can be assigned to one of two clearly distinct types, calledkappa (“κ”) and lambda (“λ”), based on the amino acid sequences of theirconstant domains.

The term IgG “isotype” or “subclass” as used herein is meant any of thesubclasses of immunoglobulins defined by the chemical and antigeniccharacteristics of their constant regions. Depending on the amino acidsequences of the constant domains of their heavy chains, antibodies(immunoglobulins) can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, γ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known and described generally in, for example, Abbas et al.Cellular and Mol. Immunology, 4th ed., W.B. Saunders, Co., 2000. Anantibody may be part of a larger fusion molecule, formed by covalent ornon-covalent association of the antibody with one or more other proteinsor peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen. The Fab fragment contains the heavy- and light-chain variabledomains and also contains the constant domain of the light chain and thefirst constant domain (CH1) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxyterminus of the heavy chain CH1 domain including one or more cysteinesfrom the antibody hinge region. Fab′-SH is the designation herein forFab′ in which the cysteine residue(s) of the constant domains bear afree thiol group. F(ab′)₂ antibody fragments originally were produced aspairs of Fab′ fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315,1994.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat.Med. 9:129-134 (2003).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,e.g., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the invention may be made by avariety of techniques, including, for example, the hybridoma method(e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al.,Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N. Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016;Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild etal., Nature Biotechnol. 14: 845-851 (1996); Neuberger, NatureBiotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, e.g., U.S. Pat. No. 4,816,567; andMorrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Chimeric antibodies include PRIMATTZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with the antigen ofinterest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from a HVR of therecipient are replaced by residues from a HVR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g.,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody.

The expression “linear antibodies” refers to the antibodies described inZapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, theseantibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

The term “about” as used herein refers to an acceptable error range forthe respective value as determined by one of ordinary skill in the art,which will depend in part how the value is measured or determined, i.e.,the limitations of the measurement system. For example, “about” can meanwithin 1 or more than 1 standard deviations, per the practice in theart. A reference to “about” a value or parameter herein includes anddescribes embodiments that are directed to that value or parameter perse. For example, a description referring to “about X” includesdescription of “X”.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a compound”optionally includes a combination of two or more such compounds, and thelike.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

II. Protein Formulations and Preparation

The invention herein relates to formulations comprising a protein and acompound which prevents oxidation of the protein in the formulation, thecompound is of formula:

wherein R² is selected from hydrogen, hydroxyl, —COOH, and —CH₂COOH;

R³ is selected from hydrogen, hydroxyl, —COOH, —CH₂COOH, and—CH₂CHR^(3a)(NH₂); wherein R^(3a) is COOH or hydrogen; R⁴, R⁵, R⁶, andR⁷ are independently selected from hydrogen and hydroxyl; provided thatone of R², R³, R⁴, R⁵, R⁶, and R⁷ is hydroxyl; or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the compound is a compound of formula:

wherein R² and R³ are independently selected from hydrogen, hydroxyl,—COOH, and —CH₂COOH; and R⁴, R⁵, R⁶, and R⁷ are independently selectedfrom hydrogen and hydroxyl; provided that one of R², R³, R⁴, R⁵, R⁶, andR⁷ is hydroxyl; or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of formula:

wherein R^(3a) is COOH or hydrogen; R², R⁴, R⁵, R⁶, and R⁷ areindependently selected from hydrogen and hydroxyl, provided that one ofR², R⁴, R⁵, R⁶, and R⁷ is hydroxyl; or a pharmaceutically acceptablesalt thereof.

In some embodiments, R⁴, R⁵ or R⁷ in any of the formula above ishydroxyl. In a further embodiment, the compound is selected from thegroup consisting of 5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxyindole, and serotonin. In some embodiments, the formulation is a liquidformulation. In some embodiments, the compound in the formulation isfrom about 0.3 mM to about 10 mM, or up to the highest concentrationthat the compound is soluble in the formulation. In some embodiments,the compound in the formulation is about 0.3 mM to about 1 mM. In someembodiments, the compound prevents oxidation of one or more amino acidsin the protein selected from group consisting of tryptophan, cysteine,histidine, tyrosine, and/or methionine. In some embodiments, thecompound prevents oxidation of the protein by a reactive oxygen species(ROS). In a further embodiment, the reactive oxygen species is selectedfrom the group consisting of a singlet oxygen, a superoxide (O₂—), analkoxyl radical, a peroxyl radical, a hydrogen peroxide (H₂O₂), adihydrogen trioxide (H₂O₃), a hydrotrioxy radical (HO₃.), ozone (O₃), ahydroxyl radical, and an alkyl peroxide. In some embodiments, a proteindescribed herein is susceptible to oxidation. In some embodiments,methionine, cysteine, histidine, tryptophan, and/or tyrosine in theprotein is susceptible to oxidation. In some embodiments, tryptophanand/or methionine in the protein is susceptible to oxidation. Forexample, a tryptophan amino acid in the Fab portion of a monoclonalantibody and/or a methionine amino acid in the Fc portion of amonoclonal antibody can be susceptible to oxidation. In someembodiments, the protein is a therapeutic protein. In some of theembodiments herein, the protein is an antibody. In some embodiments, theantibody is a polyclonal antibody, a monoclonal antibody, a humanizedantibody, a human antibody, a chimeric antibody, or an antibodyfragment. In a further embodiment, the compound prevents oxidation ofone or more amino acids in the Fab portion of an antibody. In anotherfurther embodiment, the compound prevents oxidation of one or more aminoacids in the Fc portion of an antibody. In some embodiments, theformulation provided herein is a pharmaceutical formulation suitable foradministration to a subject. As used herein a “subject” or an“individual” for purposes of treatment or administration refers to anyanimal classified as a mammal, including humans, domestic and farmanimals, and zoo, sports, or pet animals, such as dogs, horses, cats,cows, etc. Preferably, the mammal is human. In some embodiments, theformulation is aqueous. In some embodiments herein, the protein (e.g.,the antibody) concentration in the formulation is about 1 mg/mL to about250 mg/mL. In some embodiments, the formulation further one or moreexcipients selected from the group consisting of a stabilizer, a buffer,a surfactant, and a tonicity agent. For example, a formulation of theinvention can comprise a monoclonal antibody, a compound as providedherein which prevents oxidation of the protein (e.g., 5-hydroxy indole),and a buffer that maintains the pH of the formulation to a desirablelevel. In some embodiments, a formulation provided herein has a pH ofabout 4.5 to about 7.0.

Proteins and antibodies in the formulation may be prepared using methodsknown in the art. The antibody (e.g., full length antibodies, antibodyfragments and multispecific antibodies) in the formulation is preparedusing techniques available in the art, non-limiting exemplary methods ofwhich are described in more detail in the following sections. Themethods herein can be adapted by one of skill in the art for thepreparation of formulations comprising other proteins such aspeptide-based inhibitors. See Molecular Cloning: A Laboratory Manual(Sambrook et al., 4^(th) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N. Y., 2012); Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., 2003); Short Protocols in Molecular Biology(Ausubel et al., eds., J. Wiley and Sons, 2002); Current Protocols inProtein Science, (Horswill et al., 2006); Antibodies, A LaboratoryManual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manualof Basic Technique and Specialized Applications (R. I. Freshney, 6^(th)ed., J. Wiley and Sons, 2010) for generally well understood and commonlyemployed techniques and procedures for the production of therapeuticproteins, which are all incorporated herein by reference in theirentirety.

A. Antibody Preparation

The antibody in the formulations provided herein is directed against anantigen of interest. Preferably, the antigen is a biologically importantpolypeptide and administration of the antibody to a mammal sufferingfrom a disorder can result in a therapeutic benefit in that mammal.However, antibodies directed against nonpolypeptide antigens are alsocontemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule(e.g. receptor) or ligand such as a growth factor. Exemplary antigensinclude molecules such as vascular endothelial growth factor (VEGF);CD20; ox-LDL; ox-ApoB100; renin; a growth hormone, including humangrowth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; a tumor necrosis factorreceptor such as death receptor 5 and CD120; tumor necrosis factor-alphaand -beta; enkephalinase; RANTES (regulated on activation normallyT-cell expressed and secreted); human macrophage inflammatory protein(MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; receptors for hormonesor growth factors; protein A or D; rheumatoid factors; a neurotrophicfactor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3,-4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor suchas NGF-β; platelet-derived growth factor (PDGF); fibroblast growthfactor such as aFGF and bFGF; epidermal growth factor (EGF);transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des (1-3)-IGF-I (brain IGF-I),insulin-like growth factor binding proteins; CD proteins such as CD3,CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;superoxide dismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides.

(i) Antigen Preparation

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

(ii) Certain Antibody-Based Methods

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

Monoclonal antibodies of interest can be made using the hybridoma methodfirst described by Kohler et al., Nature, 256:495 (1975), and furtherdescribed, e.g., in Hongo et al., Hybridoma, 14 (3): 253-260 (1995),Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory Press, 2nd ed. 1988); Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas 563-681 (Elsevier, N. Y., 1981), andNi, Xiandai Mianyixue, 26(4):265-268 (2006) regarding human-humanhybridomas. Additional methods include those described, for example, inU.S. Pat. No. 7,189,826 regarding production of monoclonal human naturalIgM antibodies from hybridoma cell lines. Human hybridoma technology(Trioma technology) is described in Vollmers and Brandlein, Histologyand Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein,Methods and Findings in Experimental and Clinical Pharmacology,27(3):185-91 (2005).

For various other hybridoma techniques, see, e.g., US 2006/258841; US2006/183887 (fully human antibodies), US 2006/059575; US 2005/287149; US2005/100546; US 2005/026229; and U.S. Pat. Nos. 7,078,492 and 7,153,507.An exemplary protocol for producing monoclonal antibodies using thehybridoma method is described as follows. In one embodiment, a mouse orother appropriate host animal, such as a hamster, is immunized to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization. Antibodiesare raised in animals by multiple subcutaneous (sc) or intraperitoneal(ip) injections of a polypeptide of interest or a fragment thereof, andan adjuvant, such as monophosphoryl lipid A (MPL)/trehalosedicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton,Mont.). A polypeptide of interest (e.g., antigen) or a fragment thereofmay be prepared using methods well known in the art, such as recombinantmethods, some of which are further described herein. Serum fromimmunized animals is assayed for anti-antigen antibodies, and boosterimmunizations are optionally administered. Lymphocytes from animalsproducing anti-antigen antibodies are isolated. Alternatively,lymphocytes may be immunized in vitro.

Lymphocytes are then fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. See, e.g.,Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986). Myeloma cells may be used that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Exemplary myeloma cells include, but are not limited to, murinemyeloma lines, such as those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium, e.g., a medium that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells. Preferably, serum-free hybridoma cell culturemethods are used to reduce use of animal-derived serum such as fetalbovine serum, as described, for example, in Even et al., Trends inBiotechnology, 24(3), 105-108 (2006).

Oligopeptides as tools for improving productivity of hybridoma cellcultures are described in Franek, Trends in Monoclonal AntibodyResearch, 111-122 (2005). Specifically, standard culture media areenriched with certain amino acids (alanine, serine, asparagine,proline), or with protein hydrolyzate fractions, and apoptosis may besignificantly suppressed by synthetic oligopeptides, constituted ofthree to six amino acid residues. The peptides are present at millimolaror higher concentrations.

Culture medium in which hybridoma cells are growing may be assayed forproduction of monoclonal antibodies that bind to an antibody describedherein. The binding specificity of monoclonal antibodies produced byhybridoma cells may be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoadsorbent assay (ELISA). The binding affinity of the monoclonalantibody can be determined, for example, by Scatchard analysis. See,e.g., Munson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.See, e.g., Goding, supra. Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, hybridomacells may be grown in vivo as ascites tumors in an animal. Monoclonalantibodies secreted by the subclones are suitably separated from theculture medium, ascites fluid, or serum by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography. One procedure for isolation of proteins fromhybridoma cells is described in US 2005/176122 and U.S. Pat. No.6,919,436. The method includes using minimal salts, such as lyotropicsalts, in the binding process and preferably also using small amounts oforganic solvents in the elution process.

(iii) Certain Library Screening Methods

Antibodies described herein can be made by using combinatorial librariesto screen for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are describedgenerally in Hoogenboom et al. in Methods in Molecular Biology 178:1-37(O'Brien et al., ed., Human Press, Totowa, N.J., 2001). For example, onemethod of generating antibodies of interest is through the use of aphage antibody library as described in Lee et al., J. Mol. Biol. (2004),340(5):1073-93.

In principle, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the non-bindingclones in the library. The binding clones are then eluted from theantigen, and can be further enriched by additional cycles of antigenadsorption/elution. Any of the antibodies can be obtained by designing asuitable antigen screening procedure to select for the phage clone ofinterest followed by construction of a full length antibody clone usingthe Fv sequences from the phage clone of interest and suitable constantregion (Fc) sequences described in Kabat et al., Sequences of Proteinsof Immunological Interest, Fifth Edition, NIH Publication 91-3242,Bethesda Md. (1991), vols. 1-3.

In certain embodiments, the antigen-binding domain of an antibody isformed from two variable (V) regions of about 110 amino acids, one eachfrom the light (VL) and heavy (VH) chains, that both present threehypervariable loops (HVRs) or complementarity-determining regions(CDRs). Variable domains can be displayed functionally on phage, eitheras single-chain Fv (scFv) fragments, in which VH and VL are covalentlylinked through a short, flexible peptide, or as Fab fragments, in whichthey are each fused to a constant domain and interact non-covalently, asdescribed in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Asused herein, scFv encoding phage clones and Fab encoding phage clonesare collectively referred to as “Fv phage clones” or “Fv clones.”

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

In certain embodiments, filamentous phage is used to display antibodyfragments by fusion to the minor coat protein pIII. The antibodyfragments can be displayed as single chain Fv fragments, in which VH andVL domains are connected on the same polypeptide chain by a flexiblepolypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol.,222: 581-597 (1991), or as Fab fragments, in which one chain is fused topIII and the other is secreted into the bacterial host cell periplasmwhere assembly of a Fab-coat protein structure which becomes displayedon the phage surface by displacing some of the wild type coat proteins,e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137(1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-antigen clones is desired, the subject is immunizedwith antigen to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In one embodiment, a human antibodygene fragment library biased in favor of anti-antigen clones is obtainedby generating an anti-antigen antibody response in transgenic micecarrying a functional human immunoglobulin gene array (and lacking afunctional endogenous antibody production system) such that antigenimmunization gives rise to B cells producing human antibodies againstantigen. The generation of human antibody-producing transgenic mice isdescribed below.

Additional enrichment for anti-antigen reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing antigen-specific membrane bound antibody, e.g., by cellseparation using antigen affinity chromatography or adsorption of cellsto fluorochrome-labeled antigen followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which antigenis not antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). In certain embodiments, library diversity is maximized by usingPCR primers targeted to each V-gene family in order to amplify allavailable VH and VL arrangements present in the immune cell nucleic acidsample, e.g. as described in the method of Marks et al., J. Mol. Biol.,222: 581-597 (1991) or as described in the method of Orum et al.,Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplifiedDNA into expression vectors, rare restriction sites can be introducedwithin the PCR primer as a tag at one end as described in Orlandi et al.(1989), or by further PCR amplification with a tagged primer asdescribed in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities of about 10⁻⁹ M or less.

Screening of the libraries can be accomplished by various techniquesknown in the art. For example, antigen can be used to coat the wells ofadsorption plates, expressed on host cells affixed to adsorption platesor used in cell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other method for panning phagedisplay libraries.

The phage library samples are contacted with immobilized antigen underconditions suitable for binding at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for antigen.However, random mutation of a selected antibody (e.g. as performed insome affinity maturation techniques) is likely to give rise to manymutants, most binding to antigen, and a few with higher affinity. Withlimiting antigen, rare high affinity phage could be competed out. Toretain all higher affinity mutants, phages can be incubated with excessbiotinylated antigen, but with the biotinylated antigen at aconcentration of lower molarity than the target molar affinity constantfor antigen. The high affinity-binding phages can then be captured bystreptavidin-coated paramagnetic beads. Such “equilibrium capture”allows the antibodies to be selected according to their affinities ofbinding, with sensitivity that permits isolation of mutant clones withas little as two-fold higher affinity from a great excess of phages withlower affinity. Conditions used in washing phages bound to a solid phasecan also be manipulated to discriminate on the basis of dissociationkinetics.

Anti-antigen clones may be selected based on activity. In certainembodiments, the invention provides anti-antigen antibodies that bind toliving cells that naturally express antigen or bind to free floatingantigen or antigen attached to other cellular structures. Fv clonescorresponding to such anti-antigen antibodies can be selected by (1)isolating anti-antigen clones from a phage library as described above,and optionally amplifying the isolated population of phage clones bygrowing up the population in a suitable bacterial host; (2) selectingantigen and a second protein against which blocking and non-blockingactivity, respectively, is desired; (3) adsorbing the anti-antigen phageclones to immobilized antigen; (4) using an excess of the second proteinto elute any undesired clones that recognize antigen-bindingdeterminants which overlap or are shared with the binding determinantsof the second protein; and (5) eluting the clones which remain adsorbedfollowing step (4). Optionally, clones with the desiredblocking/non-blocking properties can be further enriched by repeatingthe selection procedures described herein one or more times.

DNA encoding hybridoma-derived monoclonal antibodies or phage display Fvclones is readily isolated and sequenced using conventional procedures(e.g. by using oligonucleotide primers designed to specifically amplifythe heavy and light chain coding regions of interest from hybridoma orphage DNA template). Once isolated, the DNA can be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of the desired monoclonal antibodies in therecombinant host cells. Review articles on recombinant expression inbacteria of antibody-encoding DNA include Skerra et al., Curr. Opinionin Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs, 130: 151(1992).

DNA encoding the Fv clones can be combined with known DNA sequencesencoding heavy chain and/or light chain constant regions (e.g. theappropriate DNA sequences can be obtained from Kabat et al., supra) toform clones encoding full or partial length heavy and/or light chains.It will be appreciated that constant regions of any isotype can be usedfor this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. An Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In certain embodiments,an Fv clone derived from human variable DNA is fused to human constantregion DNA to form coding sequence(s) for full- or partial-length humanheavy and/or light chains.

DNA encoding anti-antigen antibody derived from a hybridoma can also bemodified, for example, by substituting the coding sequence for humanheavy- and light-chain constant domains in place of homologous murinesequences derived from the hybridoma clone (e.g. as in the method ofMorrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNAencoding a hybridoma- or Fv clone-derived antibody or fragment can befurther modified by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In this manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of the Fv clone or hybridomaclone-derived antibodies.

(iv) Humanized and Human Antibodies

Various methods for humanizing non-human antibodies are known in theart. For example, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one embodiment of the method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Human antibodies in the formulations and compositions described hereincan be constructed by combining Fv clone variable domain sequence(s)selected from human-derived phage display libraries with known humanconstant domain sequence(s) as described above. Alternatively, humanmonoclonal antibodies can be made by the hybridoma method. Human myelomaand mouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies have been described, for example, by Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

It is possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993);and Duchosal et al. Nature 355:258 (1992).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

(v) Antibody Fragments

Antibody fragments may be generated by traditional means, such asenzymatic digestion, or by recombinant techniques. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors. For areview of certain antibody fragments, see Hudson et al. (2003) Nat. Med.9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

(vi) Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent epitopes, where the epitopes are usually from differentantigens. While such molecules normally will only bind two differentepitopes (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is typical to have thefirst heavy-chain constant region (CH1) containing the site necessaryfor light chain binding, present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. One interface comprises at least a part of the C_(H)3 domain ofan antibody constant domain. In this method, one or more small aminoacid side chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al, J. Immunol, 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60(1991).

(vii) Single-Domain Antibodies

In some embodiments, an antibody is a single-domain antibody. Asingle-domain antibody is a single polypeptide chain comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). In oneembodiment, a single-domain antibody consists of all or a portion of theheavy chain variable domain of an antibody.

(viii) Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

(ix) Antibody Derivatives

The antibodies in the formulations and compositions of the invention canbe further modified to contain additional nonproteinaceous moieties thatare known in the art and readily available. In certain embodiments, themoieties suitable for derivatization of the antibody are water solublepolymers. Non-limiting examples of water soluble polymers include, butare not limited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

(x) Vectors, Host Cells, and Recombinant Methods

Antibodies may also be produced using recombinant methods. Forrecombinant production of an anti-antigen antibody, nucleic acidencoding the antibody is isolated and inserted into a replicable vectorfor further cloning (amplification of the DNA) or for expression. DNAencoding the antibody may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(a) Signal Sequence Component

An antibody in the formulations and compositions described herein may beproduced recombinantly not only directly, but also as a fusionpolypeptide with a heterologous polypeptide, which is preferably asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. The heterologoussignal sequence selected preferably is one that is recognized andprocessed (e.g., cleaved by a signal peptidase) by the host cell. Forprokaryotic host cells that do not recognize and process a nativeantibody signal sequence, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin IIleaders. For yeast secretion the native signal sequence may besubstituted by, e.g., the yeast invertase leader, a factor leader(including Saccharomyces and Kluyveromyces α-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in WO 90/13646. In mammalian cell expression, mammalian signalsequences as well as viral secretory leaders, for example, the herpessimplex gD signal, are available.

(b) Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ, plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter.

(c) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upantibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS),thymidine kinase, metallothionein-I and -II, preferably primatemetallothionein genes, adenosine deaminase, ornithine decarboxylase,etc.

For example, cells transformed with the DHFR gene are identified byculturing the transformants in a culture medium containing methotrexate(Mtx), a competitive antagonist of DHFR. Under these conditions, theDHFR gene is amplified along with any other co-transformed nucleic acid.A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFRactivity (e.g., ATCC CRL-9096) may be used.

Alternatively, cells transformed with the GS gene are identified byculturing the transformants in a culture medium containing L-methioninesulfoximine (Msx), an inhibitor of GS. Under these conditions, the GSgene is amplified along with any other co-transformed nucleic acid. TheGS selection/amplification system may be used in combination with theDHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody of interest, wild-type DHFR gene, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(d) Promoter Component

Expression and cloning vectors generally contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding an antibody. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, β-lactamase and lactose promoter systems,alkaline phosphatase promoter, a tryptophan (trp) promoter system, andhybrid promoters such as the tac promoter. However, other knownbacterial promoters are suitable. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding an antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells can becontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(e) Enhancer Element Component

Transcription of a DNA encoding an antibody by higher eukaryotes isoften increased by inserting an enhancer sequence into the vector. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. See alsoYaniv, Nature 297:17-18 (1982) on enhancing elements for activation ofeukaryotic promoters. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the antibody-encoding sequence, but is preferablylocated at a site 5′ from the promoter.

(f) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(g) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fusion proteins, and antibody fragmentscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) that by itself showseffectiveness in tumor cell destruction. Full length antibodies havegreater half-life in circulation. Production in E. coli is faster andmore cost efficient. For expression of antibody fragments andpolypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et.al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523(Simmons et al.), which describes translation initiation region (TIR)and signal sequences for optimizing expression and secretion. See alsoCharlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression ofantibody fragments in E. coli. After expression, the antibody may beisolated from the E. coli cell paste in a soluble fraction and can bepurified through, e.g., a protein A or G column depending on theisotype. Final purification can be carried out similar to the processfor purifying antibody expressed e.g., in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger. For a reviewdiscussing the use of yeasts and filamentous fungi for the production oftherapeutic proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414(2004).

Certain fungi and yeast strains may be selected in which glycosylationpathways have been “humanized,” resulting in the production of anantibody with a partially or fully human glycosylation pattern. See,e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describinghumanization of the glycosylation pathway in Pichia pastoris); andGerngross et al., supra.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to theinvention, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also beutilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498,6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technologyfor producing antibodies in transgenic plants).

Vertebrate cells may be used as hosts, and propagation of vertebratecells in culture (tissue culture) has become a routine procedure.Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2). Other useful mammalian host cell lines include Chinese hamsterovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al., Proc. Natl.Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NSO andSp2/0. For a review of certain mammalian host cell lines suitable forantibody production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003),pp. 255-268.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(h) Culturing the Host Cells

The host cells used to produce an antibody may be cultured in a varietyof media. Commercially available media such as Ham's F10 (Sigma),Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), andDulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be usedas culture media for the host cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas GENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

(xi) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography being among one of thetypically preferred purification steps. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

In general, various methodologies for preparing antibodies for use inresearch, testing, and clinical are well-established in the art,consistent with the above-described methodologies and/or as deemedappropriate by one skilled in the art for a particular antibody ofinterest.

B. Selecting Biologically Active Antibodies

Antibodies produced as described above may be subjected to one or more“biological activity” assays to select an antibody with beneficialproperties from a therapeutic perspective. The antibody may be screenedfor its ability to bind the antigen against which it was raised. Forexample, for an anti-DR5 antibody (e.g., drozitumab), the antigenbinding properties of the antibody can be evaluated in an assay thatdetects the ability to bind to a death receptor 5 (DR5).

In another embodiment, the affinity of the antibody may be determined bysaturation binding; ELISA; and/or competition assays (e.g. RIA's), forexample.

Also, the antibody may be subjected to other biological activity assays,e.g., in order to evaluate its effectiveness as a therapeutic. Suchassays are known in the art and depend on the target antigen andintended use for the antibody.

To screen for antibodies which bind to a particular epitope on theantigen of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al., J.Biol. Chem. 270:1388-1394 (1995), can be performed to determine whetherthe antibody binds an epitope of interest.

C. Preparation of the Formulations

Provided herein are methods of preparing a formulation comprising aprotein and a compound which prevents oxidation of the protein in theformulation. The formulation may be prepared by mixing the proteinhaving the desired degree of purity with a compound which preventsoxidation of the protein in the formulation (such as a liquidformulation). In certain embodiments, the protein to be formulated hasnot been subjected to prior lyophilization and the formulation ofinterest herein is an aqueous formulation. In some embodiments, theprotein is a therapeutic protein. In certain embodiments, the protein isan antibody. In further embodiments, the antibody is a polyclonalantibody, a monoclonal antibody, a humanized antibody, a human antibody,a chimeric antibody, or antibody fragment. In certain embodiments, theantibody is a full length antibody. In one embodiment, the antibody inthe formulation is an antibody fragment, such as an F(ab′)₂, in whichcase problems that may not occur for the full length antibody (such asclipping of the antibody to Fab) may need to be addressed. Thetherapeutically effective amount of protein present in the formulationis determined by taking into account the desired dose volumes andmode(s) of administration, for example. From about 1 mg/mL to about 250mg/mL, from about 10 mg/mL to about 250 mg/mL, from about 15 mg/mL toabout 225 mg/mL, from about 20 mg/mL to about 200 mg/mL, from about 25mg/mL to about 175 mg/mL, from about 25 mg/mL to about 150 mg/mL, fromabout 25 mg/mL to about 100 mg/mL, from about 30 mg/mL to about 100mg/mL or from about 45 mg/mL to about 55 mg/mL is an exemplary proteinconcentration in the formulation. In some embodiments, the proteindescribed herein is susceptible to oxidation. In some embodiments, oneor more of the amino acids selected from the group consisting ofmethionine, cysteine, histidine, tryptophan, and tyrosine in the proteinis susceptible to oxidation. In some embodiments, tryptophan in theprotein is susceptible to oxidation. In some embodiments, methionine inthe protein is susceptible to oxidation. In some embodiments, anantibody provided herein is susceptible to oxidation in the Fab portionand/or the Fc portion of the antibody. In some embodiments, an antibodyprovided herein is susceptible to oxidation at a tryptophan amino acidin the Fab portion of the antibody. In a further embodiment, thetryptophan amino acid susceptible to oxidation is in a CDR of theantibody. In some embodiments, an antibody provided herein issusceptible to oxidation at a methionine amino acid in the Fc portion ofthe antibody.

The formulations provided herein comprise a protein and a compound whichprevents oxidation of the protein in the formulation, wherein thecompound is of formula:

wherein R² is selected from hydrogen, hydroxyl, —COOH, and —CH₂COOH;

R³ is selected from hydrogen, hydroxyl, —COOH, —CH₂COOH, and—CH₂CHR^(3a)(NH₂); wherein R^(3a) is COOH or hydrogen; R⁴, R⁵, R⁶, andR⁷ are independently selected from hydrogen and hydroxyl; provided thatone of R², R³, R⁴, R⁵, R⁶, and R⁷ is hydroxyl; or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the compound is a compound of formula:

wherein R² and R³ are independently selected from hydrogen, hydroxyl,—COOH, and —CH₂COOH; and R⁴, R⁵, R⁶, and R⁷ are independently selectedfrom hydrogen and hydroxyl; provided that one of R², R³, R⁴, R⁵, R⁶, andR⁷ is hydroxyl; or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of formula:

wherein R^(3a) is COOH or hydrogen; R², R⁴, R⁵, R⁶, and R⁷ areindependently selected from hydrogen and hydroxyl, provided that one ofR², R⁴, R⁵, R⁶, and R⁷ is hydroxyl; or a pharmaceutically acceptablesalt thereof.

In some embodiments, R⁴, R⁵ or R⁷ in any of the formula above ishydroxyl. In a further embodiment, the compound is selected from thegroup consisting of 5-hydroxy-tryptophan, 5-hydroxy indole, 7-hydroxyindole, and serotonin. In a further embodiment, the compound is selectedfrom the group consisting of 4-hydroxy indole, 5-hydroxy indole-3-aceticacid, and 7-hydroxy indole-2-carboxylic acid. In some embodiments, theformulation is a liquid formulation. In some embodiments, the compoundin the formulation is at a concentration from about 0.3 mM to about 10mM, or up to the highest concentration that the compound is soluble inthe formulation. In certain embodiments, the compound in the formulationis at a concentration from about 0.3 mM to about 9 mM, from about 0.3 mMto about 8 mM, from about 0.3 mM to about 7 mM, from about 0.3 mM toabout 6 mM, from about 0.3 mM to about 5 mM, from about 0.3 mM to about4 mM, from about 0.3 mM to about 3 mM, from about 0.3 mM to about 2 mM,from about 0.5 mM to about 2 mM, from about 0.6 mM to about 1.5 mM, orfrom about 0.8 mM to about 1.25 mM. In some embodiments, the compound inthe formulation is about 1 mM. In some embodiments, the compoundprevents oxidation of one or more amino acids in the protein. In someembodiments, the compound prevents oxidation of one or more amino acidsin the protein selected from group consisting of tryptophan, methionine,tyrosine, histidine, and/or cysteine. In some embodiments, the compoundprevents oxidation of the protein by a reactive oxygen species (ROS). Ina further embodiment, the reactive oxygen species is selected from thegroup consisting of a singlet oxygen, a superoxide (O₂—), an alkoxylradical, a peroxyl radical, a hydrogen peroxide (H₂O₂), a dihydrogentrioxide (H₂O₃), a hydrotrioxy radical (HO₃.), ozone (O₃), a hydroxylradical, and an alkyl peroxide. In a further embodiment, the compoundprevents oxidation of one or more amino acids in the Fab portion of anantibody. In another further embodiment, the compound prevents oxidationof one or more amino acids in the Fc portion of an antibody.

In some embodiments, the formulation (such as a liquid formulation)further comprises one or more excipients selected from the groupconsisting of a stabilizer, a buffer, a surfactant, and a tonicityagent. In some embodiments, the formulation is prepared in a pH-bufferedsolution. The buffer of this invention has a pH in the range from about4.5 to about 7.0. In certain embodiments the pH is in the range from pH4.5 to 6.5, in the range from pH 4.5 to 6.0, in the range from pH 4.5 to5.5, in the range from pH 4.5 to 5.0, in the range from pH 5.0 to 7.0,in the range from pH 5.5 to 7.0, in the range from pH 5.7 to 6.8, in therange from pH 5.8 to 6.5, in the range from pH 5.9 to 6.5, in the rangefrom pH 6.0 to 6.5, or in the range from pH 6.2 to 6.5. In certainembodiments of the invention, the formulation has a pH of 6.2 or about6.2. In certain embodiments of the invention, the formulation has a pHof 6.0 or about 6.0. Examples of buffers that will control the pH withinthis range include organic and inorganic acids and salts thereof. Forexample, acetate (e.g., histidine acetate, arginine acetate, sodiumacetate), succinate (e.g., histidine succinate, arginine succinate,sodium succinate), gluconate, phosphate, fumarate, oxalate, lactate,citrate, and combinations thereof. The buffer concentration can be fromabout 1 mM to about 600 mM, depending, for example, on the buffer andthe desired isotonicity of the formulation. In certain embodiments, theformulation comprises a histidine buffer (e.g., in the concentrationfrom about 5 mM to 100 mM). Examples of histidine buffers includehistidine chloride, histidine acetate, histidine phosphate, histidinesulfate, histidine succinate, etc. In certain embodiments, theformulation comprises histidine and arginine (e.g., histidinechloride-arginine chloride, histidine acetate-arginine acetate,histidine phosphate-arginine phosphate, histidine sulfate-argininesulfate, histidine succinate-arginine succinate, etc.). In certainembodiments, the formulation comprises histidine in the concentrationfrom about 5 mM to 100 mM and the arginine is in the concentration of 50mM to 500 mM. In one embodiment, the formulation comprises histidineacetate (e.g., about 20 mM)-arginine acetate (e.g., about 150 mM). Incertain embodiments, the formulation comprises histidine succinate(e.g., about 20 mM)-arginine succinate (e.g., about 150 mM). In certainembodiments, histidine in the formulation from about 10 mM to about, 35mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mMto about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 35 mM,about 20 mM to about 35 mM, about 20 mM to about 30 mM or about 20 mM toabout 25 mM. In further embodiments, the arginine in the formulation isfrom about 50 mM to about 500 mM (e.g., about 100 mM, about 150 mM, orabout 200 mM).

The formulation (such as a liquid formulation) of the invention canfurther comprise a saccharide, such as a disaccharide (e.g., trehaloseor sucrose). A “saccharide” as used herein includes the generalcomposition (CH₂O)n and derivatives thereof, including monosaccharides,disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducingsugars, nonreducing sugars, etc. Examples of saccharides herein includeglucose, sucrose, trehalose, lactose, fructose, maltose, dextran,glycerin, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol,mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose,maltose, lactulose, maltulose, glucitol, maltitol, lactitol,iso-maltulose, etc.

A surfactant can optionally be added to the formulation (such as aliquid formulation). Exemplary surfactants include nonionic surfactantssuch as polysorbates (e.g. polysorbates 20, 80, etc.) or poloxamers(e.g. poloxamer 188, etc.). The amount of surfactant added is such thatit reduces aggregation of the formulated antibody and/or minimizes theformation of particulates in the formulation and/or reduces adsorption.For example, the surfactant may be present in the formulation in anamount from about 0.001% to about 0.5%, from about 0.005% to about 0.2%,from about 0.01% to about 0.1%, or from about 0.02% to about 0.06%, orabout 0.03% to about 0.05%. In certain embodiments, the surfactant ispresent in the formulation in an amount of 0.04% or about 0.04%. Incertain embodiments, the surfactant is present in the formulation in anamount of 0.02% or about 0.02%. In one embodiment, the formulation doesnot comprise a surfactant.

In one embodiment, the formulation contains the above-identified agents(e.g., antibody, buffer, saccharide, and/or surfactant) and isessentially free of one or more preservatives, such as benzyl alcohol,phenol, m-cresol, chlorobutanol and benzethonium Cl. In anotherembodiment, a preservative may be included in the formulation,particularly where the formulation is a multidose formulation. Theconcentration of preservative may be in the range from about 0.1% toabout 2%, preferably from about 0.5% to about 1%. One or more otherpharmaceutically acceptable carriers, excipients or stabilizers such asthose described in Remington's Pharmaceutical Sciences 16th edition,Osol, A. Ed. (1980) may be included in the formulation provided thatthey do not adversely affect the desired characteristics of theformulation. Exemplary pharmaceutically acceptable excipients hereinfurther include interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

The formulation may further comprise metal ion chelators. Metal ionchelators are well known by those of skill in the art and include, butare not necessarily limited to aminopolycarboxylates, EDTA(ethylenediaminetetraacetic acid), EGTA (ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid), NTA(nitrilotriacetic acid), EDDS (ethylene diamine disuccinate), PDTA(1,3-propylenediaminetetraacetic acid), DTPA(diethylenetriaminepentaacetic acid), ADA (beta-alaninediacetic acid),MGCA (methylglycinediacetic acid), etc. Additionally, some embodimentsherein comprise phosphonates/phosphonic acid chelators.

Tonicity agents are present to adjust or maintain the tonicity of liquidin a composition. When used with large, charged biomolecules such asproteins and antibodies, they may also serve as “stabilizers” becausethey can interact with the charged groups of the amino acid side chains,thereby lessening the potential for inter- and intra-molecularinteractions. Tonicity agents can be present in any amount between 0.1%to 25% by weight, or more preferably between 1% to 5% by weight, takinginto account the relative amounts of the other ingredients. Preferredtonicity agents include polyhydric sugar alcohols, preferably trihydricor higher sugar alcohols, such as glycerin, erythritol, arabitol,xylitol, sorbitol and mannitol.

The formulation herein may also contain more than one protein or a smallmolecule drug as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect the other protein. For example, where the antibody is anti-DR5(e.g., drozitumab), it may be combined with another agent (e.g., achemotherapeutic agent, and anti-neoplastic agent).

In some embodiments, the formulation is for in vivo administration. Insome embodiments, the formulation is sterile. The formulation may berendered sterile by filtration through sterile filtration membranes. Thetherapeutic formulations herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.The route of administration is in accordance with known and acceptedmethods, such as by single or multiple bolus or infusion over a longperiod of time in a suitable manner, e.g., injection or infusion bysubcutaneous, intravenous, intraperitoneal, intramuscular,intraarterial, intralesional or intraarticular routes, topicaladministration, inhalation or by sustained release or extended-releasemeans.

The formulation of the invention may be stored in liquid or non-liquidformulation (e.g., lyophilized). The lyophilized formulation may bereconstituted before administration. In some embodiments, theconcentrations of proteins, compounds and other excipients describedherein refer to concentrations in reconstituted formulations. In someembodiments, the formulation is stable upon storage. In someembodiments, the protein in the liquid formulation is stable uponstorage at about 0 to 5° C. for at least about 12 months, at least about18 months, at least about 21 months, or at least about 24 months (or atleast about 52 weeks). In some embodiments, the physical stability,chemical stability, or biological activity of the protein in theformulation is evaluated or measured. Any methods known the art may beused to evaluate the stability and biological activity. In someembodiments, the stability is measured by oxidation of the protein inthe formulation (such as a liquid formulation) after storage. Stabilitycan be tested by evaluating physical stability, chemical stability,and/or biological activity of the antibody in the formulation around thetime of formulation as well as following storage. Physical and/orstability can be evaluated qualitatively and/or quantitatively in avariety of different ways, including evaluation of aggregate formation(for example using size exclusion chromatography, by measuringturbidity, and/or by visual inspection); by assessing chargeheterogeneity using cation exchange chromatography or capillary zoneelectrophoresis; amino-terminal or carboxy-terminal sequence analysis;mass spectrometric analysis; SDS-PAGE analysis to compare reduced andintact antibody; peptide map (for example tryptic or LYS-C) analysis;evaluating biological activity or antigen binding function of theantibody; etc. Instability may result in aggregation, deamidation (e.g.Asn deamidation), oxidation (e.g. Trp oxidation), isomerization (e.g.Asp isomeriation), clipping/hydrolysis/fragmentation (e.g. hinge regionfragmentation), succinimide formation, unpaired cysteine(s), N-terminalextension, C-terminal processing, glycosylation differences, etc. Insome embodiments, the oxidation in a protein is determined using one ormore of RP-HPLC, LC/MS, or tryptic peptide mapping. In some embodiments,the oxidation in an antibody is determined as a percentage using one ormore of RP-HPLC, LC/MS, or tryptic peptide mapping and the formula of:

${\% \mspace{14mu} {Fab}\mspace{14mu} {Oxidation}} = {100 \times \frac{{Oxidized}\mspace{14mu} {Fab}\mspace{14mu} {Peak}\mspace{14mu} {Area}}{{{Fab}\mspace{14mu} {Peak}\mspace{14mu} {Area}} + {{Oxidized}\mspace{14mu} {Fab}\mspace{14mu} {Peak}\mspace{14mu} {Area}}}}$${\% \mspace{14mu} {Fc}\mspace{14mu} {Oxidation}} = {100 \times \frac{{Oxidized}\mspace{14mu} {Fc}\mspace{14mu} {Peak}\mspace{14mu} {Area}}{{{Fc}\mspace{14mu} {Peak}\mspace{14mu} {Area}} + {{Oxidized}\mspace{14mu} {Fc}\mspace{14mu} {Peak}\mspace{14mu} {Area}}}}$

The formulations to be used for in vivo administration should besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to, or following, preparation of theformulation.

Also provided herein are methods of making a protein formulation orpreventing oxidation of a protein in a protein formulation comprisingadding an amount of a compound that prevents oxidation of a protein tothe protein formulation, wherein the compound is of formula:

wherein R² is selected from hydrogen, hydroxyl, —COOH, and —CH₂COOH;

R³ is selected from hydrogen, hydroxyl, —COOH, —CH₂COOH, and—CH₂CHR^(3a)(NH₂); wherein R^(3a) is COOH or hydrogen; R⁴, R⁵, R⁶, andR⁷ are independently selected from hydrogen and hydroxyl; provided thatone of R², R³, R⁴, R⁵, R⁶, and R⁷ is hydroxyl; or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the compound is a compound of formula:

wherein R² and R³ are independently selected from hydrogen, hydroxyl,—COOH, and —CH₂COOH; and R⁴, R⁵, R⁶, and R⁷ are independently selectedfrom hydrogen and hydroxyl; provided that one of R², R³, R⁴, R⁵, R⁶, andR⁷ is hydroxyl; or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of formula:

wherein R^(3a) is COOH or hydrogen; R², R⁴, R⁵, R⁶, and R⁷ areindependently selected from hydrogen and hydroxyl, provided that one ofR², R⁴, R⁵, R⁶, and R⁷ is hydroxyl; or a pharmaceutically acceptablesalt thereof.

In some embodiments, R⁴, R⁵ or R⁷ is hydroxyl. In some embodiments, thecompound is selected from the group consisting of 5-hydroxy-tryptophan,5-hydroxy indole, 7-hydroxy indole, and serotonin. In certainembodiments, the formulation comprises an antibody. The amount of thecompound that prevents oxidation of the protein as provided herein isfrom about 0.3 mM to about 10 mM or any of the amounts disclosed herein.

III. Administration of Protein Formulations

The formulation (such as a liquid formulation) is administered to amammal in need of treatment with the protein (e.g., an antibody),preferably a human, in accord with known methods, such as intravenousadministration as a bolus or by continuous infusion over a period oftime, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. In one embodiment, the liquid formulationis administered to the mammal by intravenous administration. For suchpurposes, the formulation may be injected using a syringe or via an IVline, for example. In one embodiment, the liquid formulation isadministered to the mammal by subcutaneous administration.

The appropriate dosage (“therapeutically effective amount”) of theprotein will depend, for example, on the condition to be treated, theseverity and course of the condition, whether the protein isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the protein, the type ofprotein used, and the discretion of the attending physician. The proteinis suitably administered to the patient at one time or over a series oftreatments and may be administered to the patient at any time fromdiagnosis onwards. The protein may be administered as the sole treatmentor in conjunction with other drugs or therapies useful in treating thecondition in question. As used herein the term “treatment” refers toboth therapeutic treatment and prophylactic or preventative measures.Those in need of treatment include those already with the disorder aswell as those in which the disorder is to be prevented. As used herein a“disorder” is any condition that would benefit from treatment including,but not limited to, chronic and acute disorders or diseases includingthose pathological conditions which predispose the mammal to thedisorder in question.

In a pharmacological sense, in the context of the invention, a“therapeutically effective amount” of a protein (e.g., an antibody)refers to an amount effective in the prevention or treatment of adisorder for the treatment of which the antibody is effective. As ageneral proposition, the therapeutically effective amount of the proteinadministered will be in the range of about 0.1 to about 50 mg/kg ofpatient body weight whether by one or more administrations, with thetypical range of protein used being about 0.3 to about 20 mg/kg,preferably about 0.3 to about 15 mg/kg, administered daily, for example.However, other dosage regimens may be useful. For example, a protein canbe administered at a dose of about 100 or 400 mg every 1, 2, 3, or 4weeks or is administered a dose of about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15.0,or 20.0 mg/kg every 1, 2, 3, or 4 weeks. The dose may be administered asa single dose or as multiple doses (e.g., 2 or 3 doses), such asinfusions. The progress of this therapy is easily monitored byconventional techniques.

IV. Methods of Screening for Compounds for the Prevention of ProteinOxidation

Also provided herein are methods of screening a compound that preventsoxidation of a protein in a protein composition. In some embodiments,the method comprises selecting a compound that has lower oxidationpotential and less photosensitivity as compared to L-tryptophan, andtesting the effect of the selected compound on preventing oxidation ofthe protein. In some embodiments, the photosensitivity is measured basedon the amount of H₂O₂ produced by the compound upon light exposure. Forexample, a liquid composition comprising the compound can be exposed to250 W/m² light for a certain amount of time and the resulting H₂O₂formation is quantified. A compound with less photosensitivity producesless H₂O₂ upon exposure to a certain amount of light than a compoundthat has a higher photosensitivity upon exposure to the same amount oflight. In some embodiments, the compound that produces less than about10%, less than about 15%, less than about 20%, less than about 25% ofthe amount of H₂O₂ is selected. H₂O₂ can be produced by oxidation ofamino acid residues in a protein that are susceptible to oxidation. Insome embodiments, the oxidation potential is measured by cyclicvoltammetry.

In some embodiments, the selected compound is tested for the effect onpreventing oxidation of the protein by reactive oxygen species generatedby 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), light, and/or aFenton reagent. In any of the embodiments herein, a method described inthe Examples may be used for screening a compound that preventsoxidation of a protein in a protein composition.

V. Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided comprising a container which holds the formulation of theinvention and optionally provides instructions for its use. Suitablecontainers include, for example, bottles, vials and syringes. Thecontainer may be formed from a variety of materials such as glass orplastic. An exemplary container is a 3-20 cc single use glass vial.Alternatively, for a multidose formulation, the container may be 3-100cc glass vial. The container holds the formulation and the label on, orassociated with, the container may indicate directions for use. Thearticle of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1 The Antioxidant L-Trp Produces ROS that Oxidize MonoclonalAntibodies in Protein Formulations

Monoclonal antibodies have been shown to produce ROS through theantibody catalyzed water oxidation pathway (ACWOP) wherein antibodiespotentially catalyze a reaction between water and singlet oxygengenerating hydrogen peroxide (Wentworth et al., Science293(5536):1806-11 (2001); Wentworth et al., Proc Natl Acad Sci USA97(20):10930-5 (2000)). In the ACWOP, a variety of ROS, includingsuperoxide anion, dihydrogen trioxide, ozone, and even hydrotrioxyradical are generated in the pathway toward production of hydrogenperoxide (Zhu et al., Proc Natl Acad Sci USA 101(8):2247-52 (2004)). Ithas been shown that surface exposed tryptophans in a monoclonal anti-DR5antibody, drozitumab (CAS number 912628-39-8), also referred to hereinas mAb1, act as substrate (¹O₂ and O₂ ⁻) generators that facilitateACWOP even under mild light conditions in a time and concentrationdependent manner (Sreedhara et al., Mol. Pharmaceutics (2013)). It wasdemonstrated that mAb1 was particularly susceptible to oxidation duringstorage under pharmaceutically relevant conditions (Sreedhara et al.,Mol. Pharmaceutics (2013)). Oxidation was shown to be site specific andlocalized to Trp53 (W53) on the heavy chain CDR (Fab) as evaluated bytryptic peptide mapping. Additionally, a reverse-phase HPLC assay wasused to measure the total oxidation in the HC Fab and Fc regions of mAb1via a papain digestion, DTT reduction, and reverse-phase separation.Peaks from RP-HPLC were identified using LC/MS and showed a strongcorrelation with results of the tryptic peptide map, indicating that theRP method could be used as a surrogate for detection of W53 (i.e. % Fab)oxidation. In the RP papain digest method, Fab and Fc oxidation peakseluted before their respective main peaks, allowing the quantificationof % Fab and % Fc oxidation in relation to their total peak areas. Thestudy further showed that hydrogen peroxide could serve as a surrogatefor a number of ROS, including superoxide and singlet oxygen.

To determine if limited light exposure can be used as an acceleratedstress model to study protein oxidation, the same human monoclonal IgG1antibody (mAb1) was used to screen and evaluate potential antioxidants.L-tryptophan (L-Trp), an antioxidant used in protein formulations, hasbeen recently shown to be photosensitive (Igarashi et al., Anal Sci23(8):943-8 (2007)) and to have the ability to produce H₂O₂ upon lightexposure. The sensitivity of mAb1 to L-Trp under light stress wasevaluated, with and without the addition of L-methionine (L-Met) as apotential antioxidant. mAb1 was expressed in Chinese Hamster Ovary (CHO)cells and purified by a series of chromatography methods includingaffinity purification by protein A chromatography and ion-exchangechromatography. mAb1 was prepared at 5 mg/mL in a formulation of 20 mMhistidine acetate, 250 mM trehalose and 0.02% polysorbate 20 in a glassvial and with 1 mM L-Trp and various concentrations of L-Met, rangingfrom 10 mM to 100 mM, and exposed to eight hours of light at 250 W/m² inan Atlas SunTest CPS+Xenon Test Instrument (Chicago, Ill.). Controlvials were wrapped in aluminum foil and treated similarly. After lightexposure, solutions were prepared for analysis by reverse-phase HPLC.For RP-HPLC, mAb1 solution from the stress study was prepared to 1.1mg/mL in 0.1 M Tris, 4.4 mM EDTA, and 1.1 mM cysteine. 150 μL of 0.1mg/mL papain was added to 1.35 mL of the mAb1 solution before incubationat 37° C. for two hours. Following incubation, 900 μL of the solutionwas combined with 100 μL of 1 M dithiothreitol (DTT) and incubated foranother thirty minutes at 37° C. Samples were then run on an Agilent,Inc. 1100/1200 HPLC system (Santa Clara, Calif.) equipped with UVdetection at 280 nm in conjunction with a Varian, Inc. Pursuit 3 μm, 2mm ID×250 mm diphenyl column (Palo Alto, Calif.). Mobile Phase A was0.1% TFA in water. Mobile Phase B was 0.1% TFA in acetonitrile. Themobile phase gradient increased linearly from 34% B at 0 minutes to 43%B at 50.0 minutes, then to 95% B at 50.1 minutes. The gradient remainedat 95% B until 60.1 minutes, and then decreased linearly from 95% B to34% B between 60.1 and 60.2 minutes. The gradient remained at 34% Buntil the end of the cycle at 80.2 minutes. The column temperature was65° C., total flow rate was 0.2 mL/min, and injection volume of eachsample was 6 μL. Chromatograms were then integrated for quantificationof oxidation.

Analysis of the light exposure effects of L-Trp and L-Met on mAb1 Faboxidation showed that the mAb1 reference material (no light exposure)and the foil control had about 2% Fab oxidation (FIG. 1A). Since thefoil control and the reference material showed the same level of Faboxidation, it was unlikely that heat alone is causing oxidation of theFab. When mAb1 was exposed to light (“No Excipient” sample), the Faboxidation doubled to 4%. With the addition of 1 mM L-Trp, the Faboxidation increased to almost 9%, suggesting that free L-Trp wasgenerating ROS under light exposure that may have resulted in oxidationof W53 on the Fab. Further addition of 10, 25, 50, and 100 mM L-Met toformulation containing 1 mM L-Trp appeared to reduce Fab oxidationslightly, but even 100 molar excess of L-Met does not reduce Faboxidation to the level of the foil control (FIG. 1A).

Oxidation in the Fc region of mAb1 has been shown to be predominately ofMet residues Met 254 and Met 430 (Sreedhara et al., Mol. Pharmaceutics(2013)). Analysis of the light exposure effects of L-Trp and L-Met onmAb1 Fc oxidation showed that the mAb1 reference material and foilcontrol had about 8% Fc oxidation even before exposure to light (FIG.1B). Exposure to light resulted in only a minor increase in Fc oxidation(“No Excipient”) for mAb1 in formulation buffer. However, incubationwith 1 mM L-Trp resulted in over 20% oxidation at these Met sites in theFc region as seen by the RP-HPLC assay. Addition of variousconcentrations of L-Met (10, 25, 50 and 100 mM) to formulationscontaining 1 mM L-Trp reduced the amount of Fc oxidation, although even100 mM L-Met does not reduce Fc oxidation to the level of the controls(FIG. 1B).

It was previously reported that L-Trp produced H₂O₂ via superoxide ionand in a sub-stoichiometric fashion while antibodies under similarconditions were producing catalytic amounts (Wentworth et al., Science293(5536):1806-11 (2001); McCormick et al., Journal of the AmericanChemical Society 100:312-313 (1978)). To test the susceptibility of freeL-Trp under pharmaceutically relevant conditions, such as under both ICHand ambient light conditions, formulations comprising 0.32 mM to 7.5 mMof L-Trp were exposed for 3 hours at 250 W/m² UV light and about 150 klux visible light. Samples were taken and analyzed immediately via theAmplex assay to detect the amount of H₂O₂ generated under theseconditions. A large quantity of H₂O₂ was generated by free L-Trp uponlight exposure in a concentration dependent manner (FIG. 2). This H₂O₂generation was reduced greatly in the presence of 50 mM sodium azide, aknown quencher of singlet oxygen (FIG. 2). When L-Trp was incubated witha combination of 50 mM NaN₃ and 150 U superoxide dismutase (SOD) or SODalone, significant amounts of H₂O₂ were still detected in the samplesnot containing NaN₃. This indicated that, in addition to singlet oxygen,superoxide ion was also generated upon photo-irradiation that wasconverted to H₂O₂ by SOD.

While confirming the photosensitivity of free L-Trp under ICH lightconditions, the effect of ambient light that was typically seen inlaboratories was studied. Measurements using a DLM1 digital light meterin various labs indicated an average of 300 lux on a lab benchtop (withwhite fluorescent lighting), an average of 3000 lux in a laminar flowhood (with white fluorescent lighting) and about 10000 lux for awindowsill exposed to southeast sunlight. Under these conditions, L-Trpin formulation buffers containing 50 mg/mL mAb1 produced hydrogenperoxide in the micromolar range as detected using the Amplex assay(FIG. 3A). Peroxide production increased with both luminosity (300,3000, and 10000 lux) and time (1, 3, and 7 days). The protein sampleswere further analyzed using the mAb1 specific RP-HPLC assay and showedincreased heavy chain Fab oxidation corresponding to oxidation in W53with increased luminosity (FIG. 3B). At the same time, % Fc oxidation inmAb1 under these conditions increased from 5 to 40% between 300 and10000 lux, respectively. These levels of light exposure and time weredetermined to be pharmaceutically relevant for drug substance handlingunder ambient light and temperature before fill/finish operations andpotentially while inspecting drug product vials. These results supportedthat L-Trp is photosensitive and that it produces several reactiveoxygen species, including singlet oxygen, superoxide and H₂O₂ that canbe detrimental to mAb product quality and that care should be takenwhile handling and storing L-Trp containing buffers.

Example 2 Screening of Candidate Antioxidant Compounds

Tryptophan (Trp) is an electron rich amino acid that undergoes oxidativeand electrophilic addition reactions in the presence of ROS such ashydroxyl radicals and singlet oxygen. Any potential antioxidant toprotect Trp oxidation in proteins should have similar if not superiorreactivity towards these ROS. A series of compounds that were eitherbased on the L-Trp structure or have been reported to have antioxidantproperties were evaluated. Compounds screened for antioxidant ability inthis study included derivatives of tryptophan, indole, aromatic acidssuch as salicylic acid and anthranilic acid, and some vitamins. Thechemical structures of the various compounds used were based on (A)Tryptophan derivatives (B) Kynurenine (C) Indole derivatives and (D)Aromatic acid derivatives:

(A) Tryptophan Derivatives

Name R X A L-Tryptophan COOH H H 5-Hydroxy-Tryptophan COOH OH H5-Methoxy-Tryptophan COOH OCH₃ H 5-Amino-Tryptophan COOH NH₂ H5-Fluoro-Tryptophan COOH F H N-Acetyl-Tryptophan COOH H CH₃C(O)Tryptamine H H H Tryptophanamide CONH₂ H H Serotonin H OH H Melatonin HOCH₃ CH₃C(O)

(B) Kynurenine

(C) Indole Derivatives

Name Y₂ Y₃ Y₄ Y₅ Y₇ Indole H H H H H Indole-3-Acetic Acid H CH₂COOH H HH 4-Hydroxy Indole H H OH H H 5-Hydroxy Indole H H H OH H 5-HydroxyIndole-3- H CH₂COOH H OH H Acetic Acid 7-Hydroxy Indole H H H H OH7-Hydroxy Indole-2- COOH H H H OH Carboxylic Acid

(D) Aromatic Acid Derivatives

Name Z₁ Z₂ Salicylic Acid OH H 5-Hydroxy Salicylic Acid OH OHAnthranilic Acid NH₂ H 5-Hydroxy Anthranilic Acid NH₂ OH

Candidate Antioxidant Compounds Obtained from a PhotosensitivityScreening Assay.

While L-Trp may have been an effective antioxidant under certaincircumstances, its photosensitivity may limit its utility during normalprocessing without special precautions. Hence the photosensitivity ofthe above molecules was investigated and rated for their H₂O₂ generationcapability with respect to L-Trp. As a screening tool, antioxidantcandidates were exposed to light for four hours at 250 W/m² and theresulting H₂O₂ formation was quantified by the Amplexassay.Specifically, antioxidants were prepared to 1 mM in 20 mM histidineacetate buffer at pH 5.5. The 1 mM antioxidant solutions were aliquotedinto glass vials (2 mL/glass vial) and exposed to four hours of light at250 W/m² in an Atlas SunTest CPS+Xenon Test Instrument (Chicago, Ill.).Total UV dose was 90 watt-hours/square meter and total visible dose was0.22 million lux hours over the 4-hour period. Control vials werewrapped in aluminum foil and treated similarly. The amount of hydrogenperoxide generated after exposure to light was measured using theAmplex® Ultra Red Assay (Invitrogen, Carlsbad, Calif.) following themanufacturer's recommended procedure. On addition of horseradishperoxidase (HRP), the dye reacted 1:1 stoichiometrically with H₂O₂,resulting in the production of fluorescent oxidation product resorufin.In this study, fluorescence readings were obtained using a Spectra MaxM2 Micro-plate Reader (Molecular Devices, Sunnyvale, Calif.) withexcitation and emission set at 560 nm and 590 nm, respectively. FinalH₂O₂ concentrations were determined using a standard curve ranging from0 μm to 20 μm.

Analysis of hydrogen peroxide (H₂O₂) generation by tryptophanderivatives upon light exposure showed that under similar conditions oflight (corresponding to 0.22 million lux hours over a 4-hour period) andbuffer (20 mM L-His-acetate, pH 5.5), 5-hydroxy-L-tryptophan producedabout one tenth of the H₂O₂, while kynurenine produced about one fifthof the H₂O₂, when compared to L-Trp (FIG. 4A). Other tryptophanderivatives produced anywhere between 30% and 105% of the H₂O₂ producedby L-Trp. In comparison to L-Trp, Trolox (a water soluble Vitamin Ederivative) produced 123 times more H₂O₂, and pyridoxine (Vitamin B6)produced 5 times more H₂O₂ (Table 1). Indole, which has a basicstructure like L-Trp, behaved similarly to L-Trp, but indole-3-aceticacid produced twice as much H₂O₂ (FIG. 4B). The hydroxy derivatives ofindole behaved like 5-OH-L-tryptophan in that they produced negligibleamounts of H₂O₂ upon light exposure. Several biochemically relevantderivatives of L-Trp, namely tryptamine, serotonin and melatonin werealso tested. Tryptamine produced about half as much H₂O₂ as L-Trp (FIG.4A). Interestingly, serotonin (5-hydroxytryptamine) behaved much likethe 5-OH derivatives of indole and tryptophan, producing very littleH₂O₂ upon light exposure, while melatonin (N-acetyl-5-methoxytryptamine)produced less than a third of the H₂O₂ produced by L-Trp (Table 1).

TABLE 1 Hydrogen Peroxide Production Ratio between Tested Compounds andL-Trp (H₂O₂ produced by Compound)/ Compound (H₂O₂ produced by L-Trp)L-Trp 1 L-Trpamide 0.43 N-Acetyl-L-Trp 0.31 N-Acetyl-L-Trpamide 0.345-Fluoro-L-Trp 0.71 5-Hydroxy-L-Trp 0.09 5-Methoxy-DL-Trp 1.055-Amino-DL-Trp 0.29 L-Kynurenine 0.20 Trolox 122.75 Pyridoxine 5.16Indole 0.95 Indole-3-Acetic Acid 2.40 4-Hydroxyindole 0.005-Hydroxyindole −0.08 5-Hydroxyindole-3-Acetic 0.11 Acid 7-Hydroxyindole−0.03 7-Hydroxyindole-2-Carboxylic 0.15 Acid Tryptamine 0.53 Serotonin(5- 0.03 Hydroxytryptamine) Melatonin (N-Acetyl-5- 0.28Methoxytryptamine) Salicylic Acid 0.03 5-Hydroxysalicylic Acid 0.84Anthranilic Acid 2.50 5-Hydroxyanthranilic Acid 0.44

In order to understand the ROS formed during photo-irradiation, severalof the Trp derivatives in the presence of 50 mM NaN₃, a known singletoxygen quencher, were tested under light exposure as described above.All the compounds tested showed a substantial decrease in the amount ofhydrogen peroxide generated under these conditions, indicating thatsinglet oxygen was a major ROS created upon photo-irradiation of Trp andits derivatives (FIG. 5).

Other aromatic compounds such as salicylic acid and derivatives werealso tested based on their reported antioxidant properties (Baltazar etal., Curr Med Chem 18(21):3252-64 (2011)). Salicylic acid produced verylittle H₂O₂ upon light exposure while its 5-OH derivative behaved likeL-Trp (Table 1). On the other hand, anthranilic acid produced twice asmuch H₂O₂ as L-Trp but 5-OH-anthranilic acid produced half as much H₂O₂compared to L-Trp (Table 1).

Candidate Antioxidant Compounds Obtained from a CV Screening Assay.

Based on the results from the photosensitivity screening assay,compounds with aromatic ring substitutions appeared to impact the amountof hydrogen peroxide generated. Since the goal was preferentialoxidation of the excipient rather than the protein drug, excipients thathad low oxidation potentials may have served as effective antioxidants.The oxidation/reduction characteristics of the compounds wereinvestigated. Several compounds, including L-Trp and derivatives, wereevaluated for the protection of Trp oxidation in proteins using cyclicvoltammetry (CV) and rank ordered based on their oxidation potentials(Table 2). Specifically, the candidate antioxidants were dissolved indeionized water and then added to a 0.2 M lithium perchlorateelectrolyte solution. Solutions were characterized with an EG&GPrinceton Applied Research Model 264A Polarograph/Voltammeter with aModel 616 RDE Glassy Carbon Electrode as working electrode. Solutionswere scanned from −0.10 V to +1.50 V at a scan rate of either 100 or 500mV/sec. The analytical cell was purged for four minutes with nitrogenbefore scanning of each antioxidant solution. The input was a linearscan of the potential of a working electrode, and the output wasmeasurement of the resulting current. As the potential was scanned(linearly increased or decreased), electrochemically active species inthe CV cell underwent oxidation and reduction reactions at the surfaceof the working electrode that resulted in a current which wascontinuously measured. Redox reactions were characterized by sharpincreases or decreases in current (peaks). The potential at which anoxidation reaction occurred was referred to as the anodic peak potential(or oxidation potential), and the potential at which a reductionoccurred was referred to as is the cathodic peak (or reduction)potential.

The oxidation potentials of the excipients in this study ranged from0.410 to 1.080 V vs Ag/AgCl (Table 2). Under these conditions, L-Trp hadan irreversible oxidation potential of 0.938 V vs Ag/AgCl. Ninecompounds were found to have a lower oxidation potential than L-Trp,including all of the 5-OH compounds which had oxidation potentialsbetween 0.535 and 0.600 V vs Ag/AgCl. Of all the compounds tested,5-amino-DL-tryptophan had the lowest oxidation potential at 0.410 V,while the N-acetyl compounds (0.730-0.880 V), and5-methoxy-DL-tryptophan (0.890 V) were also below L-Trp. Seven compoundshad higher oxidation potential than L-Trp (Table 2). These wereindole-3-acetic acid, 5-fluoro-L-tryptophan, tryptamine,L-tryptophanamide, L-kynurenine, 5-nitro-DL-tryptophan, and salicylicacid. Salicylic acid had the highest oxidation potential in this study(1.080 V vs Ag/AgCl). All the tested compounds showed non-reversible CVindicating that once oxidized, the species did not tend to receiveelectrons and probably could not be involved in further electrochemicalreactions.

TABLE 2 Oxidation Potentials of Excipients Oxidation Potential (V vsCompound Ag/AgCl) 5-amino-DL-tryptophan 0.410 5-hydroxyindole-3-aceticacid 0.535 5-hydroxy-L-tryptophan 0.565 5-hydroxyindole 0.580 SerotoninHCl (5-hydroxytryptamine 0.600 HCl) Melatonin (N-acetyl-5- 0.730methoxytryptamine) N-acetyl-L-tryptophan 0.875N-acetyl-L-tryptophanamide 0.880 5-methoxy-DL-tryptophan 0.890L-tryptophan 0.938 Indole-3-acetic acid 0.948 5-fluoro-L-tryptophan0.965 Tryptamine HCl 1.010 L-tryptophanamide 1.015 L-kynurenine 1.0405-nitro-DL-tryptophan 1.055 Salicylic acid 1.080 Oxidation (anodic peak)potentials were measured using cyclic voltammetry with a glassy carbonworking electrode in 0.2M lithium perchlorate.

A correlation was determined between oxidation potential andlight-induced H₂O₂ generation for 16 compounds that had oxidationpotentials above and below the oxidation potential of L-Trp, and H₂O₂production levels above and below that of L-Trp (FIG. 6). Since indoleand tryptophan behaved similarly in H₂O₂ production under lightexposure, it was possible that substitutions on the C₃ position of the 5membered ring did not affect this property. However, tryptamine with a—CH₂CH₂NH₂ substitution and indole-3-acetic acid with a —CH₂COOHsubstitution at the C₃ position produced two times less and two timesmore H₂O₂, respectively, than L-Trp. These data indicated that the C₃substitutions played a role in photo-activation and peroxide generation.The C₃ substitutions did not affect the oxidation potentials of themolecules, whereas indole per se had significantly lower oxidationpotential than L-Trp under these experimental conditions. Substitutionsat the C₅ of the 6-membered aromatic ring behaved quite predictably. Ingeneral, compounds with electron donating groups such as —NH₂ and —OHhad lower oxidation potentials than their parent compounds and alsoshowed low levels of H₂O₂ production upon photo-activation (e.g.5-amino-DL-tryptophan, 5-hydroxyindole-3-acetic acid,5-hydroxy-L-tryptophan, 5-hydroxyindole, serotonin). Similarly,compounds with high oxidation potential produced more H₂O₂(5-methoxy-DL-tryptophan, L-Trp, indole-3-acetic acid,5-fluoro-L-tryptophan) under these conditions. There were exceptions tothis correlation; some compounds had high oxidation potential but didnot produce much H₂O₂ (e.g. salicylic acid and L-kynurenine) indicatingthat there were potentially other mechanisms that played an importantrole for these six membered aromatic compounds that may not have beenobserved with compounds containing the indole backbone of L-Trp. Thearea of interest was the quadrant which contained compounds with loweroxidation potential and lower H₂O₂ production upon light exposure thanL-Trp (FIG. 6, dashed box). Compounds with these two qualities wereconsidered as new candidate antioxidants because they could (1) oxidizefaster than Trp on the protein and (2) produce very little H₂O₂ duringlong term storage and/or ambient processing during drug productproduction and therefore could protect the protein from furtheroxidation under these conditions.

Example 3 Candidate Antioxidant Compounds Reduced Oxidation ofMonoclonal Antibody Formulations

Compounds that, compared to L-Trp, produced less H₂O₂ upon lighttreatment as well as those with lower oxidation potentials than L-Trpwere chosen for evaluation of for their possible antioxidant propertiesusing AAPH, light, and Fenton reaction as oxidative stress models (Table3). mAb1 was used as a model protein to evaluate the effectiveness ofselect candidate antioxidants to protect against Trp oxidation by thedifferent oxidation stress models. Each stress model produced oxidationthrough a different mechanism and therefore each was valuable in theassessment of biopharmaceutical stability. AAPH, or2,2′-Azobis(2-Amidinopropane)Dihydrochloride, is used as a stress modelto mimic alkyl peroxides potentially generated from formulationexcipients such as degraded polysorbate. Decomposition of AAPH generatesalkyl, alkoxyl, and alkyl peroxyl radicals that have been shown tooxidize amino acid residues in proteins, including methionine, tyrosine,and tryptophan residues (Ji et al., J Pharm Sci 98(12):4485-500 (2009);Chao et al., Proc Natl Acad Sci USA 94(7):2969-74 (1997)). Similarly,controlled light could be used as a stress model to mimic ambient lightexposure that drugs may experience during processing and storage.Light-induced oxidation of biopharmaceuticals was shown to proceedthrough a singlet oxygen (¹O₂) and/or superoxide anion (O₂ ⁻) mechanism(Sreedhara et al., Mol. Pharmaceutics (2013)). The Fenton reaction isalso commonly used as an oxidative stress model. This mixture of H₂O₂and Fe ions generates oxidation through a metal catalyzed, hydroxylradical mechanism (Prousek et al., Pure and Applied Chemistry79(12):2325-2338 (2007)), and is used to model metal residue fromstainless steel surfaces used in drug manufacturing and storage.

TABLE 3 Oxidation Stress Models Stress Model Mechanism Purpose AAPHAlkyl peroxides, alkyl Mimic alkyl peroxides from radical catalyzeddegraded polysorbate Light Singlet oxygen (¹O₂), Mimic ambient lightexposure superoxide anion (O₂ ⁻), during processing and storage H₂O₂Fenton (H₂O₂ + Hydroxyl radical, Mimic metal residue from Fe) metalcatalyzed stainless steel surfaces

Tryptophan (W53) oxidation on mAb1 was thoroughly characterizedpreviously using a RP-HPLC and LC-MS method (Sreedhara et al., Mol.Pharmaceutics (2013)). Briefly, mAb1 was digested with papain togenerate Heavy Chain (HC) Fab, HC Fc, and Light Chain fragments. Thefragments were reduced with DTT, and then separated and identified viaLiquid Chromatography-Mass Spectrometry (LC-MS). Oxidized versions ofthe HC Fab and HC Fc were found to elute earlier than their nativecounterparts. Comparison of area integrated under the oxidized andnative peaks was used to quantify HC Fab and Fc oxidation. In addition,LC-MS/MS peptide maps (by trypsin digestion and by Lys-C digestion)showed that oxidation of the HC Fab was primarily of a Trp residue, W53,while oxidation of the HC Fc was attributed predominantly to oxidationof two Met residues, M254 and M430. By using the papain digest RP-HPLCmethod in the present study it was possible to investigate Trp residueoxidation by quantifying HC Fab oxidation, and Met residue oxidation byquantifying HC Fc oxidation.

% Fab oxidation and % Fc oxidation were calculated as follows:

${\% \mspace{14mu} {Fab}\mspace{14mu} {Oxidation}} = {100 \times \frac{{Oxidized}\mspace{14mu} {Fab}\mspace{14mu} {Peak}\mspace{14mu} {Area}}{{{Fab}\mspace{14mu} {Peak}\mspace{14mu} {Area}} + {{Oxidized}\mspace{14mu} {Fab}\mspace{14mu} {Peak}\mspace{14mu} {Area}}}}$${\% \mspace{14mu} {Fc}\mspace{14mu} {Oxidation}} = {100 \times \frac{{Oxidized}\mspace{14mu} {Fc}\mspace{14mu} {Peak}\mspace{14mu} {Area}}{{{Fc}\mspace{14mu} {Peak}\mspace{14mu} {Area}} + {{Oxidized}\mspace{14mu} {Fc}\mspace{14mu} {Peak}\mspace{14mu} {Area}}}}$

For the mAb1 stress study, mAb1 was prepared to 5 mg/mL in a formulationof 20 mM histidine acetate, 250 mM trehalose, and 0.02% Polysorbate 20.Antioxidants were added at 1 mM. Glass vials containing theseformulations were exposed to 250 W/m² light in an Atlas SunTestCPS+Xenon Test Instrument (Chicago, Ill.). Control vials were wrapped inaluminum foil and treated similarly. After light exposure, solutionswere prepared for analysis by reverse-phase HPLC as described above.

For the mAb1 AAPH stress study, mAb1 was prepared to 4 mg/mL in aformulation of 20 mM histidine acetate, 250 mM trehalose, and 0.02%Polysorbate 20. Antioxidants were added at 1 mM. 200 μL of 10 mM AAPHwas added to 2 mL of each mAb1 solution and then incubated at 40° C. for24 hours. After incubation, each solution was buffer exchanged withformulation buffer (20 mM histidine acetate, 250 mM trehalose, and 0.02%Polysorbate 20) using a PD-10 column so that the final mAb1concentration was 2.3 mg/mL. After buffer exchange, each solution wasprepared for analysis by reverse-phase HPLC as described above.

For the mAb1 Fenton stress study, mAb1 was prepared to 3 mg/mL in aformulation of 20 mM histidine hydrochloride pH 6.0. Antioxidants wereadded at a final concentration of 1 mM. A final concentration of 0.2 mMFeCl₃ and 10 ppm H₂O₂ were added to each mAb1 solution and thenincubated at 40° C. for 3 hours. After incubation, each reaction wasquenched by addition of 100 mM L-Met and then prepared for analysis byreverse-phase HPLC as described above.

It was determined that incubation of mAb1 with AAPH for 24 hours at 40°C. resulted in 27% Fab (Trp residue) oxidation (FIG. 7A) and 47% Fc (Metresidue) oxidation (FIG. 7B). Seven excipients that had been previouslyscreened using light stress and cyclic voltammetry were incubated withmAb1 under the AAPH conditions to evaluate antioxidant capabilities. Sixof the seven compounds were found to significantly reduce AAPH-inducedFab oxidation (FIG. 7A). All six of these compounds contained the indolebackbone. Moreover, all the hydroxy derivatives tested (5-hydroxy-L-Trp,5-hydroxyindole, 7-hydroxyindole, and serotonin) reduced Fab oxidationto close to control levels (about 2%). Meanwhile, salicylic acid hadalmost no effect on Fab oxidation under AAPH stress. None of theexcipients appeared to impact the level of AAPH-induced Fc oxidation(FIG. 7B).

For the light stress study, mAb1 was exposed to 16 hours of light at 250W/m² while testing the aforementioned seven excipients (FIG. 8).Exposure of mAb1 to light (“No Excipient”) increased Fab oxidation 3.5times over the control level (“mAb1 Ref Mat”, FIG. 8A). It waspreviously shown that L-Trp could protect against Trp oxidation in themodel protein Parathyroid Hormone (PTH) (Ji et al., J Pharm Sci98(12):4485-500 (2009)). However, this study found that addition of 1 mML-Trp to mAb1 increased the Fab oxidation over 11-fold, probably throughthe production of ROS such as singlet oxygen by light-exposed L-Trp(FIG. 2). Addition of the hydroxy compounds (5-hydroxy-L-Trp,5-hydroxyindole, 7-hydroxyindole, and serotonin) protected againstlight-induced Fab oxidation, reducing Fab oxidation to near controllevels (FIG. 8A). On the other hand, salicylic acid performed similarlyto L-Tryptophanamide, increasing Fab oxidation 8-fold over the controllevel. Similar results were observed for Fc oxidation under light stress(FIG. 8B). Light exposure of mAb1 resulted in a 40% increase in Fcoxidation over the control level, whereas addition of L-Trp increased Fcoxidation to 7 times the control level. Compared to the control (noexcipient), L-Tryptophanamide and salicylic acid also resulted in moreFc oxidation. The hydroxy compounds produced similar Fc oxidation as theno excipient control potentially because they produce much fewer ROSthan L-Trp under light exposure. The light screening and NaN₃ studyresults in Example 2 showed a good correlation between the amount ofH₂O₂ generated by an excipient and Fc Met oxidation of mAb1.

The Fenton reaction, using a mixture of H₂O₂ and Fe ions, generatesoxidation through a metal catalyzed, hydroxyl radical reaction (Prouseket al., Pure and Applied Chemistry 79(12):2325-2338 (2007)). Thisreaction generated Fab, i.e. tryptophan, oxidation in mAb1. The reactionwas also carried out in the presence of select antioxidants that wereuseful against both AAPH and light induced oxidation as reported above.Data related to the antioxidant properties against Fenton mediatedreaction were analyzed using the RP-HPLC assay as described above. TheFenton reaction used 10 ppm of H₂O₂ and 0.2 mM of Fe(III). The reactionwas incubated at 40° C. for 3 hours, quenched with 100 mM L-Met andanalyzed using RP-HPLC after papain digest. All samples were the averageof three separate vials, and mAb1 control (Ref Mat) was one vial withfive independent injections on the HPLC. Under the conditions tested,the Fenton reaction produced about four times the oxidation in the Fabregion of mAb1 over the control. Most of the antioxidants tested, exceptsalicylic acid, showed similar hydroxyl radical quenching properties toL-Trp, which protected the Fab oxidation by about 25% with respect tothe no excipient case. In regards to protection against Fc oxidation,the tested excipients (other than salicylic acid) performed slightlybetter than L-Trp.

1. A liquid formulation comprising a protein and a compound whichprevents oxidation of the protein in the liquid formulation, wherein thecompound is of formula:

wherein R² is selected from hydrogen, hydroxyl, —COOH, and —CH₂COOH; R³is selected from hydrogen, hydroxyl, —COOH, —CH₂COOH, and—CH₂CHR^(3a)(NH₂); wherein R^(3a) is COOH or hydrogen; R⁴, R⁵, R⁶, andR⁷ are independently selected from hydrogen and hydroxyl; provided thatone of R², R³, R⁴, R⁵, R⁶, and R⁷ is hydroxyl; or a pharmaceuticallyacceptable salt thereof.
 2. The formulation of claim 1, wherein thecompound is a compound of formula:

wherein R² and R³ are independently selected from hydrogen, hydroxyl,—COOH, and —CH₂COOH; and R⁴, R⁵, R⁶, and R⁷ are independently selectedfrom hydrogen and hydroxyl; provided that one of R², R³, R⁴, R⁵, R⁶, andR⁷ is hydroxyl; or a pharmaceutically acceptable salt thereof.
 3. Theformulation of claim 1, wherein the compound is a compound of formula:

wherein R^(3a) is COOH or hydrogen; R², R⁴, R⁵, R⁶, and R⁷ areindependently selected from hydrogen and hydroxyl, provided that one ofR², R⁴, R⁵, R⁶, and R⁷ is hydroxyl; or a pharmaceutically acceptablesalt thereof.
 4. The formulation of claim 1, wherein R⁴, R⁵ or R⁷ ishydroxyl.
 5. The formulation of claim 1, wherein the compound isselected from the group consisting of 5-hydroxy-tryptophan, 5-hydroxyindole, 7-hydroxy indole, and serotonin.
 6. The formulation of claim 1which is a pharmaceutical formulation suitable for administration to asubject.
 7. The formulation of claim 1 which is aqueous.
 8. Theformulation of claim 1, wherein the compound in the formulation is fromabout 0.3 mM to about 1 mM.
 9. The formulation of claim 1, wherein thecompound prevents oxidation of tryptophan, cysteine, histidine,tyrosine, and/or methionine in the protein.
 10. The formulation of claim1, wherein the compound prevents oxidation of the protein by a reactiveoxygen species.
 11. The formulation of claim 10, wherein the reactiveoxygen species is selected from the group consisting of singlet oxygen,hydrogen peroxide, a hydroxyl radical, and an alkyl peroxide.
 12. Theformulation of claim 1, wherein the protein is susceptible to oxidation.13. The formulation of claim 1, wherein tryptophan in the protein issusceptible to oxidation.
 14. The formulation of claim 1, wherein theprotein is an antibody.
 15. The formulation of claim 14, wherein theantibody is a polyclonal antibody, a monoclonal antibody, a humanizedantibody, a human antibody, a chimeric antibody, or antibody fragment.16. The formulation of claim 1, wherein the protein concentration in theformulation is about 1 mg/mL to about 250 mg/mL.
 17. The formulation ofclaim 1, which further comprises one or more excipients selected fromthe group consisting of a stabilizer, a buffer, a surfactant, and atonicity agent.
 18. The formulation of claim 6, wherein the formulationhas a pH of about 4.5 to about 7.0.
 19. A method of making a proteinformulation comprising adding an amount of a compound that preventsoxidation of a protein to the protein formulation, wherein the compoundis of formula:

wherein R² is selected from hydrogen, hydroxyl, —COOH, and —CH₂COOH; R³is selected from hydrogen, hydroxyl, —COOH, —CH₂COOH, and—CH₂CHR^(3a)(NH₂); wherein R^(3a) is COOH or hydrogen; R⁴, R⁵, R⁶, andR⁷ are independently selected from hydrogen and hydroxyl; provided thatone of R², R³, R⁴, R⁵, R⁶, and R⁷ is hydroxyl; or a pharmaceuticallyacceptable salt thereof.
 20. A method of preventing oxidation of aprotein in a protein formulation comprising adding an amount of acompound that prevents oxidation of the protein to the formulation,wherein the compound is of formula:

wherein R² is selected from hydrogen, hydroxyl, —COOH, and —CH₂COOH; R³is selected from hydrogen, hydroxyl, —COOH, —CH₂COOH, and—CH₂CHR^(3a)(NH₂); wherein R^(3a) is COOH or hydrogen; R⁴, R⁵, R⁶, andR⁷ are independently selected from hydrogen and hydroxyl; provided thatone of R², R³, R⁴, R⁵, R⁶, and R⁷ is hydroxyl; or a pharmaceuticallyacceptable salt thereof.
 21. The method of claim 19, wherein thecompound is a compound of formula:

wherein R² and R³ are independently selected from hydrogen, hydroxyl,—COOH, and —CH₂COOH; and R⁴, R⁵, R⁶, and R⁷ are independently selectedfrom hydrogen and hydroxyl; provided that one of R², R³, R⁴, R⁵, R⁶, andR⁷ is hydroxyl; or a pharmaceutically acceptable salt thereof.
 22. Themethod of claim 19, wherein the compound is a compound of formula:

wherein R^(3a) is COOH or hydrogen; R², R⁴, R⁵, R⁶, and R⁷ areindependently selected from hydrogen and hydroxyl, provided that one ofR², R⁴, R⁵, R⁶, and R⁷ is hydroxyl; or a pharmaceutically acceptablesalt thereof.
 23. The method of claim 19, wherein R⁴, R⁵ or R⁷ ishydroxyl.
 24. The method of claim 19, wherein the compound is selectedfrom the group consisting of 5-hydroxy-tryptophan, 5-hydroxy indole,7-hydroxy indole, and serotonin.
 25. The method of claim 19 which is apharmaceutical formulation suitable for administration to a subject. 26.The method of claim 19 which is aqueous.
 27. The method of claim 19,wherein the compound in the formulation is from about 0.3 mM to about 1mM.
 28. The method of claim 19, wherein the compound prevents oxidationof tryptophan, cysteine, histidine, tyrosine, and/or methionine in theprotein.
 29. The method of claim 19, wherein the compound preventsoxidation of the protein by a reactive oxygen species.
 30. The method ofclaim 29, wherein the reactive oxygen species is selected from the groupconsisting of singlet oxygen, hydrogen peroxide, a hydroxyl radical, andan alkyl peroxide.
 31. The method of claim 19, wherein the protein issusceptible to oxidation.
 32. The method of claim 19, wherein tryptophanin the protein is susceptible to oxidation.
 33. The method of claim 19,wherein the protein is an antibody.
 34. The method of claim 33, whereinthe antibody is a polyclonal antibody, a monoclonal antibody, ahumanized antibody, a human antibody, a chimeric antibody, or antibodyfragment.
 35. The method of claim 19, wherein the protein concentrationin the formulation is about 1 mg/mL to about 250 mg/mL.
 36. The methodof claim 19, wherein the formulation further comprises one or moreexcipients selected from the group consisting of a stabilizer, a buffer,a surfactant, and a tonicity agent.
 37. The method of claim 26, whereinthe formulation has a pH of about 4.5 to about 7.0.
 38. A method ofscreening a compound that prevents oxidation of a protein in a proteincomposition, comprising selecting a compound that has lower oxidationpotential and less photosensitivity as compared to L-tryptophan, andtesting the effect of the selected compound on preventing oxidation ofthe protein.
 39. The method of claim 38, wherein the photosensitivity ismeasured based on the amount of H₂O₂ produced by the compound upon lightexposure.
 40. The method of claim 39, wherein the compound that producesless than about 10% of the amount of H₂O₂ produced by L-tryptophan isselected.
 41. The method of claim 38, wherein the oxidation potential ismeasured by cyclic voltammetry.
 42. The method of claim 38, wherein theselected compound is tested for the effect on preventing oxidation ofthe protein by reactive oxygen species generated by2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), light, and/or aFenton reagent.